Novel supercooling methods for preservation of biological samples

ABSTRACT

The present invention provides methods for cryopreserving a population of cells with improved cell viability. In some aspects, the method comprises contacting a population of cells with a peptoid polymer comprising one or more polar peptoid monomers, e.g., formulated in a cryoprotectant solution, and cooling the population of cells at a temperature of from 0° C. to about −20° C. for a time period of at least about 3 hours to produce a population of supercooled cells. The supercooling methods of the present invention provide excellent post-thaw cell survival and recovery. In certain embodiments, the population of cells is present in a tissue or an organ that is cryopreserved by performing the supercooling methods of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/583,895 filed Sep. 26, 2019, which is a continuation of InternationalApplication No. PCT/US2018/027095 filed on Apr. 11, 2018, which claimspriority to U.S. Provisional Application No. 62/484,704 filed on Apr.12, 2017, the disclosures of which are hereby incorporated by referencein their entirety for all purposes.

BACKGROUND OF THE INVENTION

A key bottleneck to be addressed in regenerative medicine is poorcryopreservation. As cell and tissue therapies transition to pivotaltrials and commercial manufacturing, the logistics of shipping freshstarting biological materials and final drug products has proven to be acritical hurdle. Central to the success of these therapies are systemsthat provide high and reproducible levels of survival and recovery afterprolonged storage at sub-zero temperature. Hypothermic preservation (orcryopreservation) of cell and tissue therapies is necessary for earlyphase clinical trials, long-term storage prior to administration, andfor late-phase therapies manufactured centrally and distributed over alarge patient network. Such therapies administered as a cell suspensionor grafted tissue demand the use of a cryopreservation solution asvehicle that is compatible with the cells, non-toxic to the recipient,and suitable for storage of the therapy for a sufficient time prior toadministration with high viability and function.

Cryopreservation technology is aged, toxic and ineffective with typicalcell survival post-thaw below 50% and unreliable potency, which greatlyhinders standardized shipping, batch manufacturing, scheduling, andfinal product release testing. The widespread use of cryoprotectiveagents (CPAs), such as DMSO, improve post-thaw viability ofcryopreserved biological specimens by blocking ice growth, but preventsthe realization of on-demand biobanking and advanced regenerativemedicine offerings because DMSO is toxic to the cell product andsubsequently to the treated patient. See, Sauer-Heilborn et al.,Transfusion, 44 (6):907-16 (20040; Hubel, Transfusion, 41 (5):579-580(2001). Removing DMSO post-thaw has been inefficient and recoveryremains at less than 60%. See, Calmels et al., Bone Marrow Transplant,31 (9):823-828 (2000). As a strong solvent, DMSO dissolves and leachestransfusion tubing and containers, which increases risk for cGMPprocesses. Animal and human derived serum (e.g., fetal bovine serum,FBS) as a natural product with undefined structure and composition,introduces batch-to-batch variation, additional biohazards, and isgreatly influenced by supply chain issues, and geographic restrictions.Other freezing media may contain protein that is unstable leading toreduced shelf-life, or polymers that are not chemically-defined as asingle product. The FDA seeks to restrain the use of DMSO and serum viasuitable alternatives, which are needed to enable a robustinfrastructure for highly effective and off-the-shelf cell and tissueproducts.

As such, there is a need in the art for non-toxic and hyperactive iceprevention materials and methods for preserving cell- and tissue-basedsamples with high viability and function. The present disclosuresatisfies this need and provides other advantages as well.

SUMMARY OF THE INVENTION

In some aspects, provided herein is a method for cryopreserving apopulation of cells with improved cell viability, the method comprising:

-   -   (a) contacting a population of cells with a peptoid polymer or a        salt thereof comprising one or more polar peptoid monomers; and    -   (b) cooling the population of cells to a temperature of from        0° C. to about −20° C. for a time period of at least about 3        hours to produce a population of supercooled cells,    -   wherein at least about 50% of the population of supercooled        cells survive after warming to above 0° C.

In some embodiments, the percent of the population of supercooled cellsthat survive after warming is calculated by comparing the number ofcells that survive the cryopreservation method to the starting number ofcells. In some embodiments, the percent of the population of supercooledcells that survive after warming is calculated by normalizing the numberof cells that survive the cryopreservation method to a pre-determinedcell count of non-frozen cells (e.g., the starting cell number). Incertain embodiments, the percent of the population of supercooled cellsis calculated by determining the number of cells that survive thecryopreservation method at about 1 day or less (e.g., about 4, 8, 12,16, or 20 hours) after warming.

In some embodiments, the population of cells is cooled to a temperatureof from about −5° C. to about −20° C., about −6° C. to about −20° C.,about −7° C. to about −20° C., about −10° C. to about −20° C., or about−15° C. to about −20° C. In other embodiments, the population of cellsis cooled to a temperature of from 0° C. to about −15° C., 0° C. toabout −10° C., or 0° C. to about −5° C. In further embodiments, thepopulation of cells is cooled to a temperature of from about −5° C. toabout −15° C., about −5° C. to about −10° C., about −6° C. to about −15°C., about −7° C. to about −15° C., or about −10° C. to about −15° C. Incertain embodiments, the population of cells is cooled to a temperatureof about 0° C., −1° C., −2° C., −3° C., −4° C., −5° C., −6° C., −7° C.,−8° C., −9° C., −10° C., −11° C., −12° C., −13° C., −14° C., −15° C.,−16° C., −17° C., −18° C., −19° C., or −20° C. In some embodiments, thecooled population of cells is unfrozen (e.g., in a liquid, ice-freesuspension) at the temperature.

In some embodiments, the population of cells is cooled for a time periodof at least about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56,60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, or 120hours. In other embodiments, the population of cells is cooled for atime period of from about 1 to about 10 days, about 2 to about 10 days,about 3 to about 10 days, about 4 to about 10 days, about 5 to about 10days, about 1 to about 8 days, about 2 to about 8 days, about 3 to about8 days, about 4 to about 8 days, about 5 to about 8 days, about 1 toabout 5 days, about 2 to about 5 days, about 3 to about 5 days, about 4to about 5 days, about 2 to about 4 days, about 2 to about 3 days, orabout 3 to about 4 days. In further embodiments, the population of cellsis cooled for a time period of at least about 5 days (e.g., at leastabout 6, 7, 8, 9, or 10 days). In certain embodiments, the population ofcells is cooled for a time period of about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days.

In some embodiments, at least about 50% of the population of supercooledcells survive after warming to a temperature above about 5° C., 10° C.,15° C., 20° C., 25° C., 30° C., or 35° C. In other embodiments, at leastabout 50% of the population of supercooled cells survive after warmingto a temperature of from about 1° C. to about 37° C., about 5° C. toabout 37° C., about 10° C. to about 37° C., about 15° C. to about 37°C., about 20° C. to about 37° C., about 25° C. to about 37° C., about30° C. to about 37° C., about 35° C. to about 37° C., about 1° C. toabout 35° C., about 10° C. to about 35° C., about 20° C. to about 35°C., about 30° C. to about 35° C., about 1° C. to about 30° C., about 10°C. to about 30° C., about 20° C. to about 30° C., about 1° C. to about25° C., about 10° C. to about 25° C., about 20° C. to about 25° C.,about 1° C. to about 20° C., about 10° C. to about 20° C., about 1° C.to about 15° C., about 10° C. to about 15° C., about 1° C. to about 10°C., about 5° C. to about 10° C., or about 1° C. to about 5° C. Incertain embodiments, at least about 50% of the population of supercooledcells survive after warming to a temperature of about 1° C., 2° C., 3°C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C.,13° C., 14° C., 15° C. 16° C., 17° C., 18° C., 19° C., 20° C., 21° C.,22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.,31° C., 32° C., 33° C., 34° C., 35° C., 36° C., or 37° C.

In some embodiments, at least about 55% of the population of supercooledcells survive after warming. In some embodiments, at least about 60% ofthe population of supercooled cells survive after warming. In someembodiments, at least about 65% of the population of supercooled cellssurvive after warming. In some embodiments, at least about 70% of thepopulation of supercooled cells survive after warming. In someembodiments, at least about 75% of the population of supercooled cellssurvive after warming. In some embodiments, at least about 80% of thepopulation of supercooled cells survive after warming. In someembodiments, at least about 85% of the population of supercooled cellssurvive after warming. In some embodiments, at least about 90% of thepopulation of supercooled cells survive after warming. In someembodiments, at least about 95% of the population of supercooled cellssurvive after warming.

In some embodiments, at least about 50% (e.g., at least about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the population of supercooledcells survive for at least about 1 day after warming. In otherembodiments, at least about 50% (e.g., at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%) of the population of supercooled cellssurvive for a time period of from about 1 to about 10 days, about 2 toabout 10 days, about 3 to about 10 days, about 4 to about 10 days, about5 to about 10 days, about 1 to about 8 days, about 2 to about 8 days,about 3 to about 8 days, about 4 to about 8 days, about 5 to about 8days, about 1 to about 5 days, about 2 to about 5 days, about 3 to about5 days, about 4 to about 5 days, about 2 to about 4 days, about 2 toabout 3 days, or about 3 to about 4 days after warming. In furtherembodiments, at least about 50% (e.g., at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%) of the population of supercooled cellssurvive for at least about 2 days (e.g., at least about 3, 4, 5, 6, or 7days) after warming. In certain embodiments, at least about 50% (e.g.,at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of thepopulation of supercooled cells survive for about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 daysafter warming.

In some embodiments, the improved cell viability comprises enhancedproliferation of the population of supercooled cells that survive afterwarming compared to a control population of supercooled cells. In someembodiments, the control population of supercooled cells has not beencontacted with the peptoid polymer (e.g., the control population wascontacted with cryoprotectant solution only, a solution containing DMSO,or cell culture media only). In certain embodiments, the number of cellsin the population of supercooled cells at a specific time point afterwarming (e.g., about 1, 2, 3, 4, 5, 6, 7, or more days after warming) isat least about 1-fold greater (e.g., at least about 1.5-fold, 2-fold,2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold greater) than the number of cells in the control population ofsupercooled cells at that time point. As a non-limiting example, thenumber of cells in the population of supercooled cells at about 3 daysafter warming can be at least about 1-fold greater than the number ofcells in the control population of supercooled cells. As anothernon-limiting example, the number of cells in the population ofsupercooled cells at about 6 days after warming can be at least about2-fold greater than the number of cells in the control population ofsupercooled cells.

In related aspects, provided herein is a method for cryopreserving apopulation of cells with improved cell viability, the method comprising:

-   -   (a) contacting a population of cells with a peptoid polymer or a        salt thereof comprising one or more polar peptoid monomers; and    -   (b) cooling the population of cells to a temperature of from        0° C. to about −20° C. for a time period of at least about 3        hours to produce a population of supercooled cells,    -   wherein the improved cell viability comprises enhanced        proliferation of the population of supercooled cells that        survive after warming compared to a control population of        supercooled cells.

In some embodiments, the control population of supercooled cells has notbeen contacted with the peptoid polymer (e.g., the control populationwas contacted with cryoprotectant solution only, a solution containingDMSO, or cell culture media only). In certain embodiments, the number ofcells in the population of supercooled cells at a specific time pointafter warming (e.g., about 1, 2, 3, 4, 5, 6, 7, or more days afterwarming) is at least about 1-fold greater (e.g., at least about1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, or 10-fold greater) than the number of cells in thecontrol population of supercooled cells at that time point. Furtherembodiments related to these aspects of the present invention aredescribed, e.g., in paragraphs [0007] to [0012] above.

In some embodiments, the peptoid polymer is present in an amountsufficient to reduce or inhibit ice crystal formation at the temperatureto which the population of cells is cooled. In certain instances, thepeptoid polymer is present in amount between about 100 nM and about 1000mM, e.g., between about 100 nM and about 100 mM.

In some embodiments, the population of cells comprises a tissue or anorgan. In some embodiments, the population of cells comprises primarycells. In certain embodiments, the population of cells is selected fromthe group consisting of heart cells, liver cells, lung cells, kidneycells, pancreatic cells, gastric cells, intestinal cells, muscle cells,skin cells, neural cells, blood cells, immune cells, fibroblasts,genitourinary cells, bone cells, stem cells, sperm cells, oocytes,embryonic cells, epithelial cells, endothelial cells, and a combinationthereof. Non-limiting examples of genitourinary cells include corpuscavernosum cells such as, e.g., smooth muscle corpus cavernosum cells,epithelial corpus cavernosum cells, and combinations thereof.

In some embodiments, the method further comprises:

-   -   (c) warming the population of supercooled cells to above 0° C.

In some embodiments, the peptoid polymer comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, or more polar peptoid monomers. In someembodiments, the polar peptoid monomers have independently selected sidechains comprising a hydroxyl group. In some embodiments, theindependently selected side chains are optionally substituted C₁₋₁₈hydroxyalkyl groups. In some embodiments, the C₁₋₁₈ hydroxyalkyl groupsare independently selected optionally substituted C₁₋₆ hydroxyalkylgroups. In certain instances, one or more or all of the side chains ofthe polar peptoid monomers have the following structure:

Non-limiting examples of polar peptoid monomers include:

wherein the subscript m is the number of repeat units and is between 1and 10 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In someembodiments, the repeating unit, m, can be between 1 and 2, 1 and 3, 1and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, or 1 and 10.

In some embodiments, the peptoid polymer further comprises at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more hydrophobic peptoidmonomers. Non-limiting examples of hydrophobic peptoid monomers include:

wherein the subscript m is the number of repeat units and is between 1and 10 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In someembodiments, the repeating unit, m, can be between 1 and 2, 1 and 3, 1and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, or 1 and 10.

In some embodiments, the peptoid polymer is a peptoid-peptide hybrid ora salt thereof comprising the peptoid polymer and one or more aminoacids, wherein the one or more amino acids are located at one or bothends of the peptoid polymer and/or between one or more peptoid monomers.

In some embodiments, the peptoid polymer has a structure of formula (I):

a tautomer thereof or stereoisomer thereof,

-   -   wherein:    -   each R¹ is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted alkoxy,        optionally substituted C₁₋₁₈ alkylamino, optionally substituted        C₁₋₁₈ alkylthio, optionally substituted carboxyalkyl, C₃₋₁₀        cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (C₃₋₁₀        cycloalkyl)alkyl, (heterocycloalkyl)alkyl, arylalkyl, and        heteroarylalkyl;    -   wherein at least one instance of R¹ is C₁₋₁₈ hydroxyalkyl, and        wherein any of the cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl groups is optionally and independently substituted        with one or more R³ groups;    -   each R² is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted C₁₋₁₈        alkylamino, optionally substituted C₁₋₁₈ alkylthio, and        optionally substituted carboxyalkyl;    -   each R³ is independently selected from the group consisting of        halogen, oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈        hydroxyalkyl, C₁₋₈ alkylamino, and C₁₋₈ alkylthio;    -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, acetyl, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond; and    -   the subscript n, representing the number of monomers in the        polymer, is between 2 and 50.

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, ormore instances of R¹ are independently selected optionally substitutedC₁₋₁₈ hydroxyalkyl groups. In some embodiments, the C₁₋₁₈ hydroxyalkylgroups are independently selected optionally substituted C₁₋₆hydroxyalkyl groups.

In some embodiments, one or more R¹ has a structure according to R^(1b):

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

-   -   wherein:    -   m is between 1 and 8; and    -   R³ is selected from the group consisting of H, C₁₋₈ alkyl,        hydroxyl, thiol, nitro, amine, oxo, and thioxo.

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

In some embodiments, each instance of R¹ in the peptoid polymer is aC₁₋₁₈ hydroxyalkyl group. In some embodiments, each instance of R¹ is aC₁₋₆ hydroxyalkyl group. In some embodiments, each instance of R¹ is thesame C₁₋₆ hydroxyalkyl group. In some embodiments, each instance of R¹is:

In some embodiments, each instance of R² is H.

In some embodiments, the sequence length of the peptoid polymer, n, isbetween 3 and 25. In some embodiments, the sequence length of thepeptoid polymer, n, is between 5 and 25. In some embodiments, thesequence length of the peptoid polymer, n, is between 6 and 50. In someembodiments, the sequence length of the peptoid polymer, n, is between 6and 25. In some embodiments, the sequence length of the peptoid polymer,n, is between 6 and 20. In some embodiments, the sequence length of thepeptoid polymer, n, is between 8 and 50. In some embodiments, thesequence length of the peptoid polymer, n, is between 8 and 25. In someembodiments, the sequence length of the peptoid polymer, n, is between 8and 20. In some embodiments, the sequence length of the peptoid polymer,n, is between 10 and 25.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,optionally substituted C₁₋₈ alkylamino, optionally substituted C₂₋₈alkylthio, optionally substituted C₁₋₈ carboxyalkyl, or halogen.

In some embodiments, X and Y of the peptoid polymer are taken togetherto form a covalent bond.

In some embodiments, the peptoid polymer is selected from the group ofpolymers set forth in Table 2, Table 3, Table 4, Table 5, Table 6, Table7, Table 8, Table 9, or Table 10.

In some embodiments, the peptoid polymer comprises subunits comprisingone or more first hydrophobic peptoid monomers H and one or more firstpolar peptoid monomers P arranged such that the peptoid polymer has thesequence [H_(a)P_(b)]_(n) or [P_(b)H_(a)]_(n), wherein:

-   -   the subscript a, representing the number of consecutive first        hydrophobic peptoid monomers within a subunit, is between 1 and        10;    -   the subscript b, representing the number of consecutive first        polar peptoid monomers within a subunit, is between 1 and 10;        and    -   the subscript n, representing the number of subunits within the        peptoid polymer, is between 2 and 50.

In some embodiments, the peptoid polymer further comprises substituentsX and Y such that the peptoid polymer has the sequenceX—[H_(a)P_(b)]_(n)—Y or X—[P_(b)H_(a)]_(n)—Y, wherein:

-   -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond.

In some embodiments, the subunits further comprise a second hydrophobicpeptoid monomer and/or a second polar peptoid monomer such that thepeptoid polymer has the sequence [H_(a)P_(b)H_(c)P_(d)]_(n) or[P_(b)H_(a)P_(d)H_(c)]_(n), wherein:

-   -   the subscript c, representing the number of consecutive second        hydrophobic peptoid monomers within a subunit, is between 0 and        10;    -   the subscript d, representing the number of consecutive second        polar peptoid monomers within a subunit, is between 0 and 10;        and    -   both c and d are not 0.

In some embodiments, the peptoid polymer further comprises substituentsX and Y such that the peptoid polymer has the sequenceX—[H_(a)P_(b)H_(c)P_(d)]_(n)—Y or X—[P_(b)H_(a)P_(d)H_(c)]_(n)—Y,wherein:

-   -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond.

In some embodiments, the peptoid polymer further comprises a sequence Zthat comprises one or more hydrophobic peptoid monomers and/or one ormore polar peptoid monomers, wherein Z is located before the firstsubunit, after the last subunit, and/or between one or more subunits. Insome instances, Z comprises one or more hydrophobic peptoid monomers. Inother instances, Z comprises one or more polar peptoid monomers. In yetother instances, Z comprises one or more hydrophobic peptoid monomersand one or more polar peptoid monomers.

In some embodiments, the peptoid polymer comprises: (a) subunitscomprising two first hydrophobic peptoid monomers H and two first polarpeptoid monomers P, and (b) two second hydrophobic peptoid monomerslocated at the C-terminal end of the peptoid polymer, arranged such thatthe peptoid polymer has the sequence [H₂P₂]_(n)H₂ or [P₂H₂]_(n)H₂,wherein the subscript n, representing the number of subunits within thepeptoid polymer, is between 1 and 50. In some embodiments, the peptoidpolymer comprises Compound 81.

In some embodiments, the peptoid polymer further comprises substituentsX and Y such that the peptoid polymer has the sequence X—[H₂P₂]_(n)H₂—Yor X—[P₂H₂]_(n)H₂—Y, wherein X and Y are independently selected from thegroup consisting of H, optionally substituted C₁₋₈ alkyl, optionallysubstituted C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,—NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl, optionallysubstituted C₁₋₈ alkylamino, optionally substituted C₂₋₈ alkylthio,optionally substituted C₁₋₈ carboxyalkyl, and halogen, or alternativelyX and Y are taken together to form a covalent bond. In some embodiments,n is between 1 and 10.

In some embodiments, the first and/or second hydrophobic peptoidmonomers are independently selected from the hydrophobic peptoidmonomers set forth above. In some embodiments, the peptoid polymercomprises a polar peptoid monomer having a side chain that comprises ahydroxyl group. In some embodiments, the first and/or second polarpeptoid monomers are independently selected from the polar peptoidmonomers set forth above.

In some embodiments, each of the first and/or second polar peptoidmonomers comprise a side chain that is independently selected from thegroup consisting of (C₁₋₆ alkoxy)(C₁₋₆ alkylene), (oligo[ethyleneglycol]), (4- to 10-membered heterocycloalkyl)(C₁₋₆ alkylene), and (5-to 10-membered heteroaryl)(C₁₋₆ alkylene). In some embodiments, (4- to10-membered heterocycloalkyl)(C₁₋₆ alkylene) comprises a 4-6 memberedheterocyclic ring, wherein at least one member is selected from thegroup consisting of 0 and N. In some embodiments, (4- to 10-memberedheterocycloalkyl)(C₁₋₆ alkylene) comprises a tetrahydrofuranyl oroxopyrrolidinyl moiety. In some instances, the peptoid polymer comprises

In particular instances, all of the polar peptoid monomers are

In some embodiments, the peptoid polymer comprises Compound 63, Compound76, Compound 86, or Compound 87.

In some embodiments, (5- to 10-membered heteroaryl)(C₁₋₆ alkylene)comprises a 5-6 membered aromatic ring, wherein at least one ring memberis selected from the group consisting of O and N. In some embodiments,(5- to 10-membered heteroaryl)(C₁₋₆ alkylene) comprises a furanylmoiety. In some instances, the peptoid polymer comprises

In particular instances, the peptoid polymer comprises Compound 73.

In some embodiments, the side chain comprises a methoxyethyl group. Insome instances, the peptoid polymer comprises

In particular instances, the peptoid polymer comprises Compound 62.

In some embodiments, the side chain comprises an oligo(ethylene glycol)moiety. In some embodiments, the oligo(ethylene glycol) moiety is a2-(2-(2-methoxyethoxy)ethoxy)ethyl moiety. In some instances, thepeptoid polymer comprises

In particular instances, the peptoid polymer comprises Compound 67.

In some embodiments, n is between 2 and 10. In some embodiments, a isbetween 1 and 5. In some embodiments, b is between 1 and 5. In someembodiments, a is between 1 and 3 and b is between 1 and 3. In someembodiments, c is between 0 and 5. In some embodiments, d is between 0and 5.

In some embodiments, about 10 percent of the peptoid monomers arehydrophobic. In some embodiments, about 20 percent of the peptoidmonomers are hydrophobic. In some embodiments, about 30 percent of thepeptoid monomers are hydrophobic. In some embodiments, about 40 percentof the peptoid monomers are hydrophobic. In some embodiments, about 50percent of the peptoid monomers are hydrophobic. In some embodiments,about 60 percent of the peptoid monomers are hydrophobic. In someembodiments, about 70 percent of the peptoid monomers are hydrophobic.In some embodiments, about 80 percent of the peptoid monomers arehydrophobic. In some embodiments, about 90 percent of the peptoidmonomers are hydrophobic.

In some embodiments, the peptoid polymer or peptoid-peptide hybrid is inthe form of a salt. Non-limiting examples of salts include thehydrochloride, acetate, sulfate, phosphate, maleate, citrate, mesylate,nitrate, tartrate, and gluconate salts of the peptoid polymers andpeptoid-peptide hybrids described herein.

In some embodiments, the peptoid polymer and/or peptoid-peptide hybridis formulated in a cryoprotectant solution. In some embodiments, thecryoprotectant solution further comprises a compound selected from thegroup consisting of an ionic species, a penetrating cryoprotectant, anon-penetrating cryoprotectant, an antioxidant, a cell membranestabilizing compound, an aquaporin or other channel forming compound, analcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein,dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropyleneglycol (PPG), Ficoll®, polyvinylpyrrolidone, polyvinyl alcohol,hyaluronan, formamide, a natural or synthetic hydrogel, and acombination thereof.

In some embodiments, the cryoprotectant solution further comprises analcohol selected from the group consisting of propylene glycol, ethyleneglycol, glycerol, methanol, butylene glycol, adonitol, ethanol,trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol,sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol(MPD), mannitol, inositol, dithioritol, 1,2-propanediol, and acombination thereof.

In some embodiments, the cryoprotectant solution further comprises asugar that is selected from the group consisting of a monosaccharide, adisaccharide, a polysaccharide, and a combination thereof. In someinstances, the sugar is a monosaccharide selected from the groupconsisting of glucose, galactose, arabinose, fructose, xylose, mannose,3-O-Methyl-D-glucopyranose, and a combination thereof. In otherinstances, the sugar is a disaccharide selected from the groupconsisting of sucrose, trehalose, lactose, maltose, and a combinationthereof. In still other instances, the sugar is a polysaccharideselected from the group consisting of raffinose, dextran, and acombination thereof.

In some embodiments, the cryoprotectant solution further comprises a PEGor PPG that has an average molecular weight less than about 3,000 g/mol.In particular instances, the PEG or PPG has an average molecular weightbetween about 200 and 400 g/mol.

In some embodiments, the cryoprotectant solution further comprises aprotein selected from the group consisting of bovine serum albumin,human serum albumin, gelatin, and a combination thereof. In someembodiments, the cryoprotectant solution further comprises a natural orsynthetic hydrogel that comprises chitosan, hyaluronic acid, or acombination thereof. In some embodiments, the cryoprotectant solutionfurther comprises a nonionic surfactant selected from the groupconsisting of polyoxyethylene lauryl ether, polysorbate 80, and acombination thereof.

In further aspects, provided herein is a population of supercooled cellswith improved cell viability produced by any of the methods describedherein.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general protocol for the synthesis of peptoidoligomers using the “submonomer” approach.

FIGS. 2A and 2B show the results of a capillary tube freeze assay thatwas performed at −20° C. FIG. 2A illustrates the assay in whichCompounds 1 (1 eq.) and 10 (1 eq.) were dissolved in MilliQ water andsubjected to subzero temperatures. Comparison was made to water aloneand a solution of ethylene glycol (EG) (18 eq.). FIG. 2B displaysnormalized results of the assay depicted in FIG. 2A.

FIGS. 3A-3D show x-ray diffraction (XRD) crystallography data. FIG. 3Ashows XRD data for a solution containing 5 mM Compound 12 and 17.5%(v/v) ethylene glycol (EG). FIG. 3B shows XRD data for a solutioncontaining 30% (v/v) EG. FIG. 3C shows XRD data for a solutioncontaining 17.5% (v/v) EG. FIG. 3D shows ice ring scores for a number ofsolutions containing EG, Compound 2 (labeled as “B”), Compound 12(labeled as “D”), and/or Compound 8 (labeled as “E”). For each differentsolution, two separate ice ring scores were determined.

FIGS. 4A-4G show x-ray diffraction (XRD) crystallography data forsolutions containing 5 mg/mL of Compound 10, Compound 12, Compound 8,Compound 13, Compound 11, and Compound 58, compared to an ethyleneglycol (EG) control. Each solution also contained 300 mM NaCl, 100 mMHEPES, 15% (v/v) ethylene glycol, and pH was adjusted to 7.2. FIG. 4A:Compound 10 XRD crystallography pattern (left) and spectrum plot(right). FIG. 4B: Compound 12 XRD crystallography pattern (left) andspectrum plot (right). FIG. 4C: Compound 8 XRD crystallography pattern(left) and spectrum plot (right). FIG. 4D: Compound 13 XRDcrystallography pattern (left) and spectrum plot (right). FIG. 4E:Compound 11 XRD crystallography pattern (left) and spectrum plot(right). FIG. 4F: Compound 58 XRD crystallography pattern (left) andspectrum plot (right). FIG. 4G: EG control XRD crystallography pattern(left) and spectrum plot (right). For XRD spectrum plots, intensity wasplotted as a function of angle (20 degrees).

FIGS. 5A-5G show x-ray diffraction (XRD) crystallography data forsolutions containing 1 mg/mL of Compound 10, Compound 12, Compound 8,Compound 13, Compound 11, and Compound 58, compared to an ethyleneglycol (EG) control. Each solution also contained 300 mM NaCl, 100 mMHEPES, 17.5% (v/v) ethylene glycol, and pH was adjusted to 7.2. FIG. 5A:Compound 10 XRD crystallography pattern (left) and spectrum plot(right). FIG. 5B: Compound 12 XRD crystallography pattern (left) andspectrum plot (right). FIG. 5C: Compound 8 XRD crystallography pattern(left) and spectrum plot (right). FIG. 5D: Compound 13 XRDcrystallography pattern (left) and spectrum plot (right). FIG. 5E:Compound 11 XRD crystallography pattern (left) and spectrum plot(right). FIG. 5F: Compound 58 XRD crystallography pattern (left) andspectrum plot (right). FIG. 5G: EG control XRD crystallography pattern(left) and spectrum plot (right). For XRD spectrum plots, intensity wasplotted as a function of angle (20 degrees).

FIGS. 6A-6C show two solutions that were flash frozen, rewarmed, andsubsequently refrozen. The control solution contained 22.5% (v/v)ethylene glycol (EG), while the test solution contained 22.5% EG and 5mg/mL (0.5% (w/v)) Compound 12. FIG. 6A shows that during rapid freezingin liquid nitrogen, the solution containing Compound 12 vitrified whilethe control solution completely froze. FIG. 6B shows that duringrewarming at 37° C., the solution containing Compound 12 unfroze (withintwo seconds) while the control stayed frozen. FIG. 6C shows that afterovernight in a −20° C. freezer, the Compound 12 solution remainedunfrozen, unlike the control.

FIG. 7 shows the results of a cell toxicity study performed on HEK 293cells in which Compound 12 (squares) or DMSO (circles) was added to cellculture media. A sample in which no Compound 12 or DMSO was added(“Culture Media” (triangles)) served as a control. Serial dilutions wereperformed in order to test different concentrations of Compound 12 andDMSO.

FIG. 8 shows the results of a cryopreservation assay performed on HEK293 cells, comparing a solution containing ethylene glycol (EG) to asolution containing EG and Compound 12. Cell viability was measured 12hours post-thaw.

FIG. 9 shows the results of a cryopreservation assay performed on HEK293 cells, comparing a solution containing 5 mg/mL of Compound 12 plus amixture of glycols, disaccharides, and a general buffer to solutionscontaining VS2E or M22. Cell viability was measured 16 hours post-thaw.Cells were vitrified with liquid nitrogen (LN2).

FIG. 10A shows an example of flow cytometric data collected after 72 hrstorage of Jurkat cells at −20° C. Cell number was normalized to anon-frozen control for comparison. Formulas XT-SC3 and XT-SC4 containHTK buffer, Compound 12, and other components. Both HTK buffer andDMEM+10% FBS showed little to no survival after 24 hr storage. XT-SC4showed little to no cell death after 72 hrs and XT-SC3 showed only aslight decline in cell survival as time points extended.

FIG. 10B shows a fluorescence viability assay after 72 hr storage ofHEK293 cells at −20° C., corroborating flow cytometry results withJurkat cells. Here, HTK and DMEM/FBS provided no survival at −20° C. andvery poor survival at 4° C. after 24 hrs.

FIG. 11 shows a time course survival of rat corpus cavernosum (CC)endothelial cells stored in supercooling formula (XT-SC5 to 8) and HTKsolution at −20° C. for up to 48 hrs. After treatment with supercoolingmedia, the cells were recovered into culture media (50× dilution) at 37°C. for 24 hrs prior to staining with Calcein AM. Quantitativefluorescence signaling indicates cell survival in the bar chart and thecorrelated cell images are shown on the bottom.

FIG. 12 shows the survival of SK-OV-3 cells cryopreserved for 3 days at−20° C. in either XT Formula with 1% Compound 12, XT Formula only, 10%DMSO formula, or DMEM cell media only. Cell survival was measured 1 daypost-warming.

FIG. 13 shows the proliferation of SK-OV-3 cells cryopreserved for 3days at −20° C. in either XT Formula with 1% Compound 12, 10% DMSOformula, or DMEM cell media only. Cell viability was measured 3 dayspost-warming.

FIG. 14 shows the proliferation of K562 cells cryopreserved for 3 daysat −20° C. in either XT Formula with 1% Compound 12, XT Formula only,10% DMSO formula, or DMEM cell media only. Cell viability was measured 3days post-warming.

FIG. 15 shows the proliferation of K562 cells cryopreserved for 3 daysat −20° C. in either XT Formula with 1% Compound 12, XT Formula only,10% DMSO formula, or DMEM cell media only. Cell viability was measured 6days post-warming.

FIG. 16 shows the proliferation of K562 cells cryopreserved for 3 daysat −20° C. in either XT Formula with 1% Compound 12, 27, 62, 74, or 76,10% DMSO formula, or DMEM cell media only. Cell viability was measured 7days post-warming.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The banking of cells and tissues at low temperatures usingcryopreservation is critical for many biological products andapplications, but remains a significant problem that has yet to allowthe successful full recovery or viable therapeutic cells, tissues, andorgans. Cryopreservation is typically performed with cryoprotectiveagents (CPAs), which are critical chemical additives such as dimethylsulfoxide (DMSO), bovine serum albumin (BSA), and others. The CPAs areused to improve the post-thaw viability of cryopreserved biologicalsystems by preventing ice crystal nucleation and growth. However, theseagents exhibit various levels of cytotoxicity at their effectiveconcentrations and thus limit the success of cryopreservation,biobanking, and advanced regenerative medicine. This lack of aneffective and safe CPA contributes to the widespread use of toxic CPAs.

The present invention is based, in part, on the discovery that peptoidpolymers comprising at least one polar peptoid monomer (e.g., having apolar side chain such as a hydroxyl group) can be used in ice-freesupercooling methods that provide excellent cell survival and recoverywhen cell populations are cooled to supercooling temperatures (e.g., 0°C. to −20° C.) for a time period of at least about 3 hours, compared tocontrol samples without peptoid polymers that suffer a complete loss ofcell viability after 3-24 hrs. As a non-limiting example, studies withhuman model cell types (e.g., HEK-293, K562, Jurkat, SK-OV-3) using theice-free supercooling methods described herein have demonstrated typicalpost-thaw (e.g., post-warming) cell recoveries in excess of 85%, attimes achieving nearly 100% survival 16-hours post-thaw.

II. Abbreviations and Definitions

The abbreviations used herein are conventional, unless otherwisedefined. The following abbreviations are used to refer to the monomerunits of the peptoid polymer: Nsb (2-(sec-butylamino)acetic acid), Nib(2-(isobutylamino)acetic acid), Nbu (2-butylamino)acetic acid), Npr(2-propylamino)acetic acid), Nip (2-(isopropylamino)acetic acid), Nme(2-(methylamino)acetic acid), Nhp (2-((2-hydroxypropyl)amino)aceticacid), Nhe (2-((2-hydroxyethyl)amino)acetic acid), Ndp(2-((2,3-dihydroxypropryl)amino)acetic acid, Nyp(2-((1-hydroxypropan-2-yl)amino) acetic acid), Nep(2-((1-(4-hydroxyphenyl)ethyl)amino) acetic acid, Ndh(2-((1,3,-dihydrooxypropan-2-yl)amino)acetic acid, Nop(2-((3-(2-oxopyrrolindin-1-yl)propyl)amino)acetic acid, Nmo(2-(2-methoxyethylamino)acetic acid), Ntf(2-((tetrahydrofuran-2-yl)methylamino)acetic acid), Nff(2-(furan-2-ylmethylamino)acetic acid), Nmb(2-(2-methylbutylamino)acetic acid), Nrh(2-(R)-(2-hydroxypropylamino)acetic acid), Nsh(2-(S)-(2-hydroxypropylamino)acetic acid), N3p(2-(2-(2-(2-methoxyethoxy)ethoxy)ethylamino)acetic acid), Nbr((2-(R)-sec-butylamino)acetic acid), and Nbs((2-(S)-sec-butylamino)acetic acid). The following abbreviations areused to refer to chemical compounds: DMF (N, N′-dimethylformamide), DIEA(diisopropylethylamine), DIC (N, N′-diisopropylcarbodiimide), ACN(acetonitrile), DCM (methylene chloride), HFIP (hexafluoroisopropylalcohol); Fmoc (9-fluorenylmethoxycarbonyl).

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The term “about” as used herein to modify a numerical value indicates adefined range around that value. If “X” were the value, “about X” wouldindicate a value from 0.9X to 1.1X, and more preferably, a value from0.95X to 1.05X. Any reference to “about X” specifically indicates atleast the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groupshaving up to 30 carbons atoms, such as, but not limited to heptyl,octyl, nonyl, decyl, etc. Alkyl groups can be substituted orunsubstituted. Alkyl groups can be optionally substituted with one ormore moieties selected from halo, hydroxy, amino, thiol, alkylamino,alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond. Alkenyl can includeany number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈,C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆.Alkenyl groups can have any suitable number of double bonds, including,but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groupsinclude, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substitutedor unsubstituted. Alkenyl groups can be optionally substituted with oneor more moieties selected from halo, hydroxy, amino, thiol, alkylamino,alkoxy, haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Alkynyl” refers to either a straight chain or branched hydrocarbonhaving at least 2 carbon atoms and at least one triple bond. Alkynyl caninclude any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇,C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, andC₆. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.Alkynyl groups can be substituted or unsubstituted. Alkynyl groups canbe optionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is anynumber of suitable carbon atoms. Representative alkylene groups include,but are not limited to, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylenegroups can be substituted or unsubstituted. Alkylene groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkenylene” refers to an alkenyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkenylene can be linked to the same atom ordifferent atoms of the alkenylene. Alkenylene groups include, but arenot limited to, ethenylene, propenylene, isopropenylene, butenylene,isobutenylene, sec-butenylene, pentenylene and hexenylene. Alkenylengroups can be substituted or unsubstituted. Alkenylene groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Alkynylene” refers to an alkynyl group, as defined above, linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkynylene can be linked to the same atom ordifferent atoms of the alkynylene. Alkynylene groups include, but arenot limited to, ethynylene, propynylene, isopropynylene, butynylene,sec-butynylene, pentynylene and hexynylene. Alkynylene groups can besubstituted or unsubstituted. Alkynylene groups can be optionallysubstituted with one or more moieties selected from halo, hydroxy,amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo,thioxo, and cyano.

“Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

“Amine” or “amino” refers to an —N(R)₂ group where the R groups can behydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,or heteroaryl, among others. The R groups can be the same or different.The amino groups can be primary (each R is hydrogen), secondary (one Ris hydrogen) or tertiary (each R is other than hydrogen). The alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroarylgroups can be optionally substituted with one or more moieties selectedfrom halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, thioxo, and cyano.

“Hydroxyl” or “hydroxy” refers to an —OH group. The hydroxyl can be atany suitable carbon atom.

“Thiol” refers to an —SH group. The thiol group can be at any suitablecarbon atom.

“Oxo” refers to a double bonded O group (═O, —C(O)—). The oxo group canbe at any suitable carbon atom.

“Thioxo” refers to a double bonded S group (═S). The thioxo group can beat any suitable carbon atom.

“Nitro” refers to a —NO₂ group. The nitro group can be at any suitablecarbon atom.

“Carboxy” refers to a carboxylic acid group of the formula —C(O)OH or—CO₂H.

“Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈,C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkyl ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl ringsinclude, for example, norbornane, [2.2.2] bicyclooctane,decahydronaphthalene and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈cycloalkyl, exemplary groups include, but are not limited tocyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When cycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl,exemplary groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can besubstituted or unsubstituted. Cycloalkyl groups can be optionallysubstituted with one or more moieties selected from alkyl, alkenyl,alkynyl, halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy,amido, thiol, nitro, oxo, thioxo, and cyano. For example, cycloalkylgroups can be substituted with C₁₋₆ alkyl or oxo (═O), among manyothers.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 12ring members and from 1 to 4 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized to form moietites including,but not limited to, —S(O)— and —S(O)₂—. Heterocycloalkyl groups caninclude any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ringmembers. Any suitable number of heteroatoms can be included in theheterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to4, 2 to 3, 2 to 4, or 3 to 4. The heterocycloalkyl group can includegroups such as aziridine, azetidine, pyrrolidine, piperidine, azepane,azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-,1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane(tetrahydropyran), oxepane, thiirane, thietane, thiolane(tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane,morpholine, thiomorpholine, dioxane, or dithiane. The heterocycloalkylgroups can also be fused to aromatic or non-aromatic ring systems toform members including, but not limited to, indoline. Heterocycloalkylgroups can be unsubstituted or substituted. Heterocycloalkyl groups canbe optionally substituted with one or more moieties selected from alkyl,alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. For example,heterocycloalkyl groups can be substituted with C₁₋₆ alkyl or oxo (═O),among many others.

The heterocycloalkyl groups can be linked via any position on the ring.For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

When heterocycloalkyl includes 3 to 8 ring members and 1 to 3heteroatoms, representative members include, but are not limited to,pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene,thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, morpholine,thiomorpholine, dioxane and dithiane. Heterocycloalkyl can also form aring having 5 to 6 ring members and 1 to 2 heteroatoms, withrepresentative members including, but not limited to, pyrrolidine,piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine,imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, and morpholine.

“Aryl” refers to an aromatic ring system having any suitable number ofring atoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, having a methylene linking group. Some arylgroups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted. Aryl groups canbe optionally substituted with one or more moieties selected from alkyl,alkenyl, alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Heteroaryl” refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized to form moieties including,but not limited to, —S(O)— and —S(O)₂—. Heteroaryl groups can includeany number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members.Any suitable number of heteroatoms can be included in the heteroarylgroups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from5 to 8 ring members and from 1 to 4 heteroatoms, or from 5 to 8 ringmembers and from 1 to 3 heteroatoms, or from 5 to 6 ring members andfrom 1 to 4 heteroatoms, or from 5 to 6 ring members and from 1 to 3heteroatoms. The heteroaryl group can include groups such as pyrrole,pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine,pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers),thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Theheteroaryl groups can also be fused to aromatic ring systems, such as aphenyl ring, to form members including, but not limited to,benzopyrroles such as indole and isoindole, benzopyridines such asquinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted. Heteroaryl groups can be optionallysubstituted with one or more moieties selected from alkyl, alkenyl,alkynyl, halo, hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl,carboxy, amido, nitro, oxo, thioxo, and cyano.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

“(Cycloalkyl)alkyl” refers to a radical having an alkyl component and acycloalkyl component, where the alkyl component links the cycloalkylcomponent to the point of attachment. The alkyl component is as definedabove, except that the alkyl component is at least divalent, analkylene, to link to the cycloalkyl component and to the point ofattachment. The alkyl component can include any number of carbons, suchas C₁₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅,C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. The cycloalkyl component is as definedwithin. Exemplary (cycloalkyl)alkyl groups include, but are not limitedto, methyl-cyclopropyl, methyl-cyclobutyl, methyl-cyclopentyl andmethyl-cyclohexyl.

“(Heterocycloalkyl)alkyl” refers to a radical having an alkyl componentand a heterocycloalkyl component, where the alkyl component links theheterocycloalkyl component to the point of attachment. The alkylcomponent is as defined above, except that the alkyl component is atleast divalent, an alkylene, to link to the heterocycloalkyl componentand to the point of attachment. The alkyl component can include anynumber of carbons, such as C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₂₋₃,C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. Theheterocycloalkyl component is as defined above. (Heterocycloalkyl)alkylgroups can be substituted or unsubstituted.

“Arylalkyl” refers to a radical having an alkyl component and an arylcomponent, where the alkyl component links the aryl component to thepoint of attachment. The alkyl component is as defined above, exceptthat the alkyl component is at least divalent, an alkylene, to link tothe aryl component and to the point of attachment. The alkyl componentcan include any number of carbons, such as C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅,C₁₋₆, C₂₋₃, C₂₋₄, C₂-s, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. Thearyl component is as defined above. Examples of arylalkyl groupsinclude, but are not limited to, benzyl and ethyl-benzene. Arylalkylgroups can be substituted or unsubstituted.

“Heteroarylalkyl” refers to a radical having an alkyl component and aheteroaryl component, where the alkyl component links the heteroarylcomponent to the point of attachment. The alkyl component is as definedabove, except that the alkyl component is at least divalent, analkylene, to link to the heteroaryl component and to the point ofattachment. The alkyl component can include any number of carbons, suchas C₀₋₆, C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄,C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. The heteroaryl component is as definedwithin. Heteroarylalkyl groups can be substituted or unsubstituted.

“Carboxyalkyl” refers to a carboxy group linked to an alkyl, asdescribed above, and generally having the formula —C₁₋₈ alkyl-C(O)OH.Any suitable alkyl chain is useful. Carboxyalkyl groups can beoptionally substituted with one or more moieties selected from halo,hydroxy, amino, thiol, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano.

“Acyl” refers to an alkyl that contains an oxo substituted carbon at thepoint of attachment (—C(O)—C₁₋₈ alkyl). Any suitable alkyl chain isuseful. Acyl groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, amino, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano.

“Hydroxyalkyl” refers to an alkyl group, as defined above, where atleast one of the hydrogen atoms is replaced with a hydroxy group. As forthe alkyl group, hydroxyalkyl groups can have any suitable number ofcarbon atoms, such as C₁₋₆. Exemplary hydroxyalkyl groups include, butare not limited to, hydroxy-methyl, hydroxyethyl (where the hydroxy isin the 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-,2- or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3-or 4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-,4- or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-,4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like. Hydroxyalkylgroups can be optionally substituted with one or more moieties selectedfrom halo, thiol, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido,nitro, oxo, thioxo, and cyano. One of skill in the art will appreciatethat other hydroxyalkyl groups are useful in the present invention.

“Alkoxy” refers to an alkyl group having at least one bridging oxygenatom. The bridging oxygen atom can be anywhere within the alkyl chain(alkyl-O-alkyl) or the bridging oxygen atom can connect the alkyl groupto the point of attachment (alkyl-O—). In some embodiments, the bridgingoxygen atom is not present as a terminal hydroxy group (i.e., —OH). Insome instances, the alkoxy contains 1, 2, 3, 4, or 5 bridging oxygenatoms. As for alkyl group, alkoxy groups can have any suitable number ofcarbon atoms, such as C₁₋₂, C₁₋₄, and C₁₋₆. Alkoxy groups include, forexample, methoxy, ethoxy, propoxy, iso-propoxy, methyloxy-ethyloxy-ethyl(C₁—O—C₂—O—C₂—), etc. One example of an alkoxy group is polyethyleneglycol (PEG) wherein the polyethylene glycol chain can include between 2to 20 ethylene glycol monomers. Alkoxy groups can be optionallysubstituted with one or more moieties selected from halo, hydroxy,amino, thiol, alkylamino, haloalkyl, carboxy, amido, nitro, oxo, thioxo,and cyano. Alkoxy groups can be substituted or unsubstituted.

“Alkylamino” refers to an alkyl group as defined within, having one ormore amino groups. The amino groups can be primary, secondary ortertiary. Alkylamino groups useful in the present invention include, butare not limited to, ethyl amine, propyl amine, isopropyl amine, ethylenediamine and ethanolamine. The amino group can link the alkylamino to thepoint of attachment with the rest of the compound, be at any position ofthe alkyl group, or link together at least two carbon atoms of the alkylgroup. Alkylamino groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, thiol, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skillin the art will appreciate that other alkylaminos are useful in thepresent invention.

“Alkylthio” refers to an alkyl group as defined within, having one ormore thiol groups. Alkylthio groups useful in the present inventioninclude, but are not limited to, ethyl thiol, propyl thiol, andisopropyl thiol. The thiol group can link the alkylthio to the point ofattachment with the rest of the compound, be at any position of thealkyl group, or link together at least two carbon atoms of the alkylgroup. Alkylthio groups can be optionally substituted with one or moremoieties selected from halo, hydroxy, amino, alkylamino, alkoxy,haloalkyl, carboxy, amido, nitro, oxo, thioxo, and cyano. One of skillin the art will appreciate that other alkylthio are useful in thepresent invention.

The term “oxyethyl” refers to a divalent radical having the formula—OCH₂CH₂—.

The term “wavy line” signifies the point of attachment of thesubstituent to the remainder of the molecule. When the wavy line is notdepicted as being specifically appended to a specific ring atom, thepoint of attachment can be to any suitable atom of the substituent. Forexample, the wavy line in the following structure:

is intended to include, as the point of attachment, any of thesubstitutable atoms.

The term “regenerative medicine” refers to a branch of medicine thatdeals with the process of replacing, engineering or regenerating humancells, tissues, or organs to restore or establish normal function. Insome embodiments, regenerative medicine includes growing tissues andorgans in the laboratory and safely implanting them when the body cannotheal itself.

The term “supercooling” refers to the process of cooling a substancebelow a phase-transition temperature without the transition occurring.For example, supercooling can refer to the process of lowering thetemperature of a liquid below its freezing point without solidificationor crystallization.

The term “bioengineered tissue” refers to one or more syntheticallycreated cells, tissues, or organs created for the purposes ofregenerative medicine. In some embodiments, bioengineered tissue refersto cells, tissues, or organs that were developed in the laboratory. Insome embodiments, bioengineered tissues refers to laboratory derivedheart, liver, lung, kidney, pancreas, intestine, thymus, cornea, stemcells (e.g., human pluripotent stem cells, hematopoietic stem cells),lymphocytes, granulocytes, immune system cells, bone cells, organoids,embryonic cells, oocytes, sperm cells, blood platelets, nerve cells, ora combination thereof.

The term “cryoprotectant solution” refers to a solution used to reduceor prevent freezing damage caused by ice crystal formation. In someembodiments, the cryoprotectant solution comprises one or more peptoidpolymers described herein. In other embodiments, the cryoprotectantsolution comprises one or more peptoid polymers and one or morepeptoid-peptide hybrids described herein. In some embodiments, thecryoprotectant solution protects a biological sample from freezingdamage. In some embodiments, the cryoprotectant solution protects anon-biological sample from ice crystal formation. In some embodiments,the cryoprotectant solution preserves a biological sample for an amountof time longer than if the biological sample were not exposed to reducedtemperatures.

The terms “vitrify” and “vitrification” mean the transformation of asubstance into a glass (i.e., non-crystalline amorphous solid). In thecontext of water, vitrification refers to the transformation of waterinto a glass without the formation of ice crystals, as opposed toordinary freezing, which results in ice crystal formation. Vitrificationis often achieved through very rapid cooling and/or the introduction ofagents that suppress ice crystal formation. On the other hand,“devitrify” and “devitrification” refer to the process ofcrystallization in a previously crystal-free (amorphous) glass. In thecontext of water ice, devitrification can mean the formation of icecrystals as the previously non-crystalline amorphous solid undergoesmelting.

The term “peptoid polymer” or “peptoid” refers to a polyamide of betweenabout 2 and 1,000 (e.g., between about 2 and 1,000, 2 and 950, 2 and900, 2 and 850, 2 and 800, 2 and 750, 2 and 700, 2 and 650, 2 and 600, 2and 550, 2 and 500, 2 and 450, 2 and 400, 2 and 350, 2 and 300, 2 and250, 2 and 200, 2 and 150, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 2 and 9, 2 and8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, or 2 and 3) peptoid monomershaving substituents “R¹” on the amide nitrogen atoms. Optionally, asecond, independently selected, substituent “R²” can be attached to thecarbon atom that is α- to the carbonyl group (i.e., attached to theα-carbon atom). R² can be, but is not limited to, H. In particularinstances, a peptoid is a synthetic analog of a peptide wherein the sidechains that would otherwise be attached to the α-carbon atoms areinstead attached to the amide nitrogen atoms. In general, peptoids aresynthetic polymers with controlled monomer sequences and lengths thatcan be made by automated solid-phase organic synthesis to include a widevariety of side chains having different chemical functions. R¹ groupsbonded to the amide nitrogen atoms in the peptoid monomers can include,but are not limited to, H, optionally substituted C₁₋₁₈ alkyl,optionally substituted C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈alkynyl, optionally substituted C₁₋₁₈ hydroxyalkyl, optionallysubstituted alkoxy, optionally substituted C₁₋₁₈ alkylamino, optionallysubstituted C₁₋₁₈ alkylthio, optionally substituted carboxyalkyl, C₃₋₁₀cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (C₃₋₁₀ cycloalkyl)alkyl,(heterocycloalkyl)alkyl, arylalkyl, and heteroarylalkyl groups, whereinany of the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups isoptionally and independently substituted with one or more “R³” groups.Each R³ group can be independently selected from halogen, oxo, thioxo,—OH, —SH, sulfonamide, amino, C₁₋₈ alkyl, C₁₋₈ hydroxyalkyl, C₁₋₈alkylamino, or C₁₋₈ alkylthio groups. Furthermore, R¹ groups cancomprise the side chain of any of the amino acids alanine (Ala),cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine(Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg),lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline(Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val),tryptophan (Trp), or tyrosine (Tyr). The term includes free amine formsas well as salt forms.

The terms “polar peptoid monomer” and “peptoid monomer having a polarside chain” are used interchangeably to refer to peptoid monomers inwhich the substituent “R¹” is a polar side chain, or both R¹ and thesubstituent “R²” are polar side chains. Commonly, a polar side chainwill comprise a hydroxyl group and/or an atom (e.g., sulfur, nitrogen,oxygen) that can participate in hydrogen bonding. In some instances, apolar side chain includes atoms or groups that are more hydrophobic thanpolar in nature (e.g., aromatic rings). In these instances, the sidechain also includes atoms or groups such that the entire side chain ismore polar than hydrophobic. As a non-limiting example, a polar sidechain can contain an aromatic ring to which one or more hydroxyl groupsare attached.

In some embodiments, the peptoid polymer comprises one or more polarpeptoid monomers selected from the group consisting of Nop, Nhp, Nhe,Ndp, Nyp, Nep, Ndh, and a combination thereof.

The terms “hydrophobic peptoid monomer” and “peptoid monomer having ahydrophobic side chain” are used interchangeably to refer to peptoidmonomers in which the substituent “R¹” is a hydrophobic side chain(e.g., not polar), or both IV and the substituent “R²” are hydrophobicside chains. Commonly, a hydrophobic side chain comprises anunsubstituted alkyl, unsubstituted cycloalkyl, or an unsubstitutedaromatic group.

In some embodiments, the peptoid polymer comprises one or morehydrophobic peptoid monomers selected from the group consisting of Nsb,Nib, Nbu, Npr, Nip, Nme, and a combination thereof.

The term “peptoid-peptide hybrid” refers to an oligomer that is composedof both peptoid monomer units and alpha amino acids (i.e., peptideunits). The term includes free amine forms as well as salt forms.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues, oran assembly of multiple polymers of amino acid residues.

The term “amino acid” includes but is not limited to naturally-occurringα-amino acids and their stereoisomers. “Stereoisomers” of amino acidsrefers to mirror image isomers of the amino acids, such as L-amino acidsor D-amino acids. For example, a stereoisomer of a naturally-occurringamino acid refers to the mirror image isomer of the naturally-occurringamino acid (i.e., the D-amino acid).

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified (e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine).Naturally-occurring α-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of a naturally-occurring α-aminoacids include, without limitation, D-alanine (D-Ala), D-cysteine(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu),D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile),D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine(D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln),D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan(D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. For example, an L-aminoacid may be represented herein by its commonly known three letter symbol(e.g., Arg for L-arginine) or by an upper-case one-letter amino acidsymbol (e.g., R for L-arginine). A D-amino acid may be representedherein by its commonly known three letter symbol (e.g., D-Arg forD-arginine) or by a lower-case one-letter amino acid symbol (e.g., r forD-arginine).

III. Detailed Description of the Embodiments

Provided herein are methods for cryopreserving a population of cellssuch as, e.g., cells present in a tissue or organ. In some aspects, themethod comprises contacting a population of cells with a peptoid polymercomprising one or more polar peptoid monomers, and cooling thepopulation of cells to a temperature of from 0° C. to about −20° C. fora desired time period, e.g., at least about 3 hours. The supercoolingmethods of the present invention advantageously provide excellentpost-thaw cell survival and recovery of tissues and organs suitable fortransplantation.

A. Peptoid Polymers

In some aspects, provided herein is a peptoid polymer of formula (I):

-   -   a tautomer thereof or stereoisomer thereof,    -   wherein:    -   each R¹ is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted alkoxy,        optionally substituted C₁₋₁₈ alkylamino, optionally substituted        C₁₋₁₈ alkylthio, optionally substituted carboxyalkyl, C₃₋₁₀        cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (C₃₋₁₀        cycloalkyl)alkyl, (heterocycloalkyl)alkyl, arylalkyl, and        heteroarylalkyl,    -   wherein at least one instance of R¹ is C₁₋₁₈ hydroxyalkyl, and    -   wherein any of the cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl groups is optionally and independently substituted        with one or more R³ groups;    -   each R₂ is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted C₁₋₁₈        alkylamino, optionally substituted C₁₋₁₈ alkylthio, and        optionally substituted carboxyalkyl;    -   each R³ is independently selected from the group consisting of        halogen, oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈        hydroxyalkyl, C₁₋₈ alkylamino, and C₁₋₈ alkylthio;    -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, acetyl, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond; and    -   the subscript n, representing the number of monomers in the        polymer, is between 2 and 50.

In some embodiments, all instances of R¹ are not hydroxyethyl when n isbetween 3 and 7. In some embodiments, each instance of R¹ in the peptoidpolymer is selected from the group consisting of:

wherein: m is between 1 and 8; and R³ is selected from the groupconsisting of H, C₁₋₈ alkyl, hydroxyl, thiol, nitro, amine, oxo, andthioxo. In some embodiments, the repeating unit, m, can be between 1 and2, 1 and 3, 1 and 4, 1 and 5, 1 and 6, or 1 and 7. In some embodiments,the repeating unit, m, is 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, one or more IV monomers has a structure accordingto Ria:

In some embodiments, each R^(1a) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more IV monomers has a structure accordingto Rib:

In some embodiments, each Rib group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more IV monomers has a structure accordingto R^(1c):

In some embodiments, each R^(1c) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more IV monomers has a structure accordingto Ria:

In some embodiments, each Rid group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

In some embodiments, one or more R¹ monomers has a structure accordingto R^(1e):

In some embodiments, each R^(1e) group is independently selected from

In some embodiments, a mixture of the two stereoisomers are chosen. Insome embodiments, only the R stereoisomer of the monomer is chosen. Insome embodiments, only the S stereoisomer of this monomer is chosen.

Whenever any monomer herein does not indicate stereochemistry, anystereoisomer may be used. In some embodiments, a mixture of the twostereoisomers are chosen. In embodiments comprising a mixture ofstereoisomers, the ratio of R to S stereoisomer of the monomer in thepeptoid polymer can range from about 95:5 to about 90:10, from about90:10 to about 85:15, from about 85:15 to about 80:20, from about 80:20to about 75:25, from about 75:25 to about 70:30, from about 70:30 toabout 65:35, from about 65:35 to about 60:40, from about 60:40 to about55:45, from about 55:45 to about 50:50, from about 50:50 to about 45:55,from about 45:55 to about 40:60, from about 40:60 to about 35:65, fromabout 35:65 to about 30:70, from about 30:70 to about 25:75, from about25:75 to about 20:80, from about 20:80 to about 15:85, from about 15:85to about 10:90, or from about 10:90 to about 5:95. In some embodiments,only the R stereoisomer of the monomer is chosen. In some embodiments,only the S stereoisomer of the monomer is chosen.

Whenever a particular stereochemistry is shown with a wedge or a dashedline, the monomer is substantially free of other stereoisomers. In someembodiments, substantially free means at least 70% pure. In someembodiments, substantially free means at least 80% pure. In someembodiments, substantially free means at least 90% pure. In someembodiments, substantially free means at least 95% pure. In someembodiments, substantially free means at least 99% pure. In someembodiments, substantially free means at least 99.9% pure.

In some embodiments, each instance of R¹ in the peptoid polymer isselected from the group consisting of:

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more instances of R¹ in the peptoidpolymer are independently selected C₁₋₁₈ hydroxyalkyl groups (e.g.,independently selected C₁₋₆ hydroxyalkyl groups). In some embodiments,each instance of R¹ in the peptoid polymer is a C₁₋₁₈ hydroxyalkylgroup. In some embodiments, each instance of R¹ is a C₁₋₆ hydroxyalkylgroup. In some embodiments, each instance of R¹ is the same C₁₋₆hydroxyalkyl group. In some embodiments, each instance of R¹ is anhydroxyalkyl group where the length of the alkyl in each hydroxyalkylgroup is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or more carbon atoms. In some embodiments thehydroxyalkyl group contains 1, 2, 3, 4, 5, 6, 7, or 8 hydroxysubstitutions. In some embodiments, each instance of R¹ is:

In some embodiments, each instance of R² is H. In some embodiments, atleast one R² is a halogen.

In some embodiments, the sequence length of the peptoid polymer, n, isbetween 3 and 25. In some embodiments, the sequence length of thepeptoid polymer, n, is between 5 and 25. In some embodiments, thesequence length of the peptoid polymer, n, is between 6 and 50. In someembodiments, the sequence length of the peptoid polymer, n, is between 6and 25. In some embodiments, the sequence length of the peptoid polymer,n, is between 6 and 20. In some embodiments, the sequence length of thepeptoid polymer, n, is between 8 and 50. In some embodiments, thesequence length of the peptoid polymer, n, is between 8 and 25. In someembodiments, the sequence length of the peptoid polymer, n, is between 8and 20. In some embodiments, the sequence length of the peptoid polymer,n, can be between from about 10 to about 28, from about 12 to about 26,from about 14 to about 24, from about 16 to about 22, or from about 18to about 20. In some embodiments, the sequence length of the peptoidpolymer, n, can be between from about 8 to about 50, from about 8 toabout 45, from about 8 to about 40, from about 8 to about 35, from about8 to about 30, from about 10 to about 25, from about 10 to about 20, orfrom about 10 to about 15. In some embodiments, the sequence length ofthe peptoid polymer, n, can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, acetyl, carboxy, optionally substituted C₁₋₈hydroxyalkyl, optionally substituted C₁₋₈ alkylamino, optionallysubstituted C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, orhalogen. In some embodiments, X is an acetyl group. In some embodiments,Y is carboxy.

In some embodiments, X and Y of the peptoid polymer are taken togetherto form a covalent bond. The formation of a covalent bond between X andY results in a circularized form of the peptoid polymer in which theterminal NR¹ group and the terminal C═O group are linked, as shownbelow.

In some embodiments, the peptoid polymer consists of monomer unitsselected from the group of monomers set forth in Table 1. A person ofskill in the art will recognize that the bounds of this invention arenot limited to the monomers listed in Table 1, and that any usefulN-substituted substituent can be used as an N-substituted peptoidmonomer. In some embodiments, the N-substituted substituent on theN-substituted peptoid monomer is any of the side chains of the aminoacids alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid(Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine(Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), or tyrosine (Tyr).

TABLE 1

In some embodiments, the peptoid polymer is selected from the group ofpeptoid polymers set forth in Table 2, Table 3, Table 4, Table 5, Table6, Table 7, Table 8, Table 9, or Table 10.

TABLE 2

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

TABLE 3

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

TABLE 4

Compound 23

Compound 24

TABLE 5

Compound 25

Compound 26

Compound 27

Compound 28

TABLE 6

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

TABLE 7

Compound 35

Compound 36

Compound 37

Compound 38

Compound 39

Compound 40

TABLE 8

Compound 41

Compound 42

Compound 43

Compound 44

Compound 45

Compound 46

Compound 47

Compound 48

Compound 49

TABLE 9

Compound 50

Compound 51

Compound 52

Compound 53

Compound 54

Compound 55

Compound 56

Compound 57

TABLE 10

Compound 59

Compound 60

Compound 61

Compound 62

Compound 63

Compound 64

Compound 65

Compound 66

Compound 67

Compound 68

Compound 69

Compound 70

Compound 71

Compound 72

Compound 73

Compound 74

Compound 75

Compound 76

Compound 77

Compound 78

Compound 79

Compound 80

Compound 81

Compound 82

Compound 83

Compound 84

Compound 85

Compound 86

Compound 87

Compound 88

Compound 89

Compound 90

Compound 91

Compound 92

Compound 93

Compound 94

Compound 95

Compound 96

Compound 97

Compound 98

Compound 99

Compound 100

Compound 101

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nsb monomers, 1 Nhp monomerand 9 Nsb monomers, 2 Nhp monomers and 8 Nsb monomers, 3 Nhp monomersand 7 Nsb monomers, 4 Nhp monomers and 6 Nsb monomers, 5 Nhp monomersand 5 Nsb monomers, 6 Nhp monomers and 4 Nsb monomers, 7 Nhp monomersand 3 Nsb monomers, 8 Nhp monomers and 2 Nsb monomers, 9 Nhp monomersand 1 Nsb monomer, or 10 Nhp monomers.

In some embodiments, the peptoid polymer has the sequenceNhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp, wherein X is H or C₁₋₈ acyl andY is —OH or —NH₂ or C₁₋₈alkyl. In some embodiments, the peptoid polymerhas the sequence Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb, wherein X is Hor C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments,the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp, wherein X is H or C₁₋₈ acyl andY is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, the peptoid polymerhas the sequence Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb, wherein X is Hor C₁₋₈ acyl and Y is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments,the peptoid polymer has the sequenceNsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp, wherein X is H or C₁₋₈ acyl andY is —OH or —NH₂ or C₁₋₈ alkyl. In some embodiments, Y is a secondaryamine or a tertiary amine.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nme monomers, 1 Nhp monomerand 9 Nme monomers, 2 Nhp monomers and 8 Nme monomers, 3 Nhp monomersand 7 Nme monomers, 4 Nhp monomers and 6 Nme monomers, 5 Nhp monomersand 5 Nme monomers, 6 Nhp monomers and 4 Nme monomers, 7 Nhp monomersand 3 Nme monomers, and 8 Nhp monomers and 2 Nme monomers, or 9 Nhpmonomers and 1 Nme monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 1 Nhe monomers and 9 Nsbmonomers, 2 Nhe monomers and 8 Nsb monomers, 3 Nhe monomers and 7 Nsbmonomers, 4 Nhe monomers and 6 Nsb monomers, 5 Nhe monomers and 5 Nsbmonomers, 6 Nhe monomers and 4 Nsb monomers, 7 Nhe monomers and 3 Nsbmonomers, 8 Nhe monomers and 2 Nsb monomers, 9 Nhe monomers and 1 Nsbmonomers, or 10 Nhe monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nbu monomers, 1 Nhp monomerand 9 Nbu monomers, 2 Nhp monomers and 8 Nbu monomers, 3 Nhp monomersand 7 Nbu monomers, 4 Nhp monomers and 6 Nbu monomers, 5 Nhp monomersand 5 Nbu monomers 6 Nhp monomers and 4 Nbu monomers, 7 Nhp monomers and3 Nbu monomers, 8 Nhp monomers and 2 Nbu monomers, or 9 Nhp monomers and1 Nbu monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nib monomers, 1 Nhp monomerand 9 Nib monomers, 2 Nhp monomers and 8 Nib monomers, 3 Nhp monomersand 7 Nib monomers, 4 Nhp monomers and 6 Nib monomers, 5 Nhp monomersand 5 Nib monomers, 6 Nhp monomers and 4 Nib monomers, 7 Nhp monomersand 3 Nib monomers, 8 Nhp monomers and 2 Nib monomers, or 9 Nhp monomersand 1 Nib monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Npr monomers, 1 Nhp monomerand 9 Npr monomers, 2 Nhp monomers and 8 Npr monomers, 3 Nhp monomersand 7 Npr monomers, 4 Nhp monomers and 6 Npr monomers, 5 Nhp monomersand 5 Npr monomers, 6 Nhp monomers and 4 Npr monomers, 7 Nhp monomersand 3 Npr monomers, 8 Nhp monomers and 2 Npr monomers, or 9 Nhp monomersand 1 Npr monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is10, and the peptoid polymer comprises: 10 Nip monomers, 1 Nhp monomerand 9 Nip monomers, 2 Nhp monomers and 8 Nip monomers, 3 Nhp monomersand 7 Nip monomers, 4 Nhp monomers and 6 Nip monomers, 5 Nhp monomersand 5 Nip monomers, 6 Nhp monomers and 4 Nip monomers, 7 Nhp monomersand 3 Nip monomers, 8 Nhp monomers and 2 Nip monomers, or 9 Nhp monomersand 1 Nip monomer.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nsbmonomers, 7 Nhp monomers and 7 Nsb monomers, 8 Nhp monomers and 6 Nsbmonomers, 10 Nhp monomers and 4 Nsb monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is14, and the peptoid polymer comprises: 6 Nhp monomers and 8 Nibmonomers, 7 Nhp monomers and 7 Nib monomers, 8 Nhp monomers and 6 Nibmonomers, 10 Nhp monomers and 4 Nib monomers, or 14 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is16, and the peptoid polymer comprises: 5 Nhp monomers and 11 Nsbmonomers, 7 Nhp monomers and 9 Nsb monomers, 8 Nhp monomers and 8 Nsbmonomers, 10 Nhp monomers and 6 Nsb monomers, 12 Nhp monomers and 4 Nsbmonomers, or 16 Nhp monomers.

In some embodiments, the sequence length of the peptoid polymer, n, is22, and the peptoid polymer comprises: 7 Nhp monomers and 15 Nsbmonomers, 10 Nhp monomers and 12 Nsb monomers, 11 Nhp monomers and 11Nsb monomers, 14 Nhp monomers and 8 Nsb monomers, 17 Nhp monomers and 5Nsb monomers, or 22 Nhp monomers.

In other aspects, provided herein is a peptoid polymer comprisingsubunits comprising one or more first hydrophobic peptoid monomers H andone or more first polar peptoid monomers P arranged such that thepeptoid polymer has the sequence [H_(a)P_(b)]_(n) or [P_(b)H_(a)]_(n),wherein the subscript a represents the number of consecutive firsthydrophobic peptoid monomers within a subunit, the subscript brepresents the number of consecutive first polar peptoid monomers withina subunit, and the subscript n represents the number of subunits withinthe peptoid polymer. In some embodiments, a is between 1 and 10 (e.g., ais 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In other embodiments, b is between1 and 10 (e.g., b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In someinstances, a is between 1 and 5. In other instances, b is between 1 and5. In particular instances, a is between 1 and 3 and b is between 1 and3.

In some embodiments, n is between 2 and 50 (e.g., n is 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50). In some instances, n is between 2 and 10(e.g., n is 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n isbetween 3 and 25. In some embodiments, n is between 5 and 25. In someembodiments, n is between 8 and 50. In some embodiments, n is between 8and 25. In some embodiments, n is between 8 and 20. In some embodiments,n can be between from about 10 to about 28, from about 12 to about 26,from about 14 to about 24, from about 16 to about 22, or from about 18to about 20. In some embodiments, n can be between from about 8 to about50, from about 8 to about 45, from about 8 to about 40, from about 8 toabout 35, from about 8 to about 30, from about 10 to about 25, fromabout 10 to about 20, or from about 10 to about 15.

In some embodiments, the sequence length of the peptoid polymer isbetween 6 and 50. In some embodiments, the sequence length of thepeptoid polymer is between 10 and 50. In some embodiments, the sequencelength of the peptoid polymer is between 16 and 100. In someembodiments, the sequence length of the peptoid polymer is between 16and 50. In some embodiments, the sequence length of the peptoid polymeris between 16 and 40. In some embodiments, the sequence length of thepeptoid polymer can be between from about 20 to about 56, from about 24to about 52, from about 28 to about 48, from about 32 to about 44, orfrom about 36 to about 40. In some embodiments, the sequence length ofthe peptoid polymer can be between from about 16 to about 100, fromabout 16 to about 90, from about 16 to about 80, from about 16 to about70, from about 16 to about 60, from about 20 to about 50, from about 20to about 40, or from about 20 to about 30. In some embodiments, thesequence length of the peptoid polymer can be 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, ormore.

When more than one hydrophobic peptoid monomer is present in a peptoidpolymer, all of the hydrohpbic peptoid monomers can be the same, theycan all be different, or a combination thereof. Similarly, when morethan one polar peptoid monomer is present in a peptoid polymer, all ofthe polar peptoid monomers can be the same, they can all be different,or a combination thereof.

In some embodiments, the subunits further comprise a second hydrophobicpeptoid monomer and/or a second polar peptoid monomer such that thepeptoid polymer has the sequence [H_(a)P_(b)H_(c)P_(d)]_(n) or[P_(b)H_(a)P_(d)H_(c)]_(n), wherein the subscript c represents thenumber of consecutive second hydrophobic peptoid monomers within asubunit and the subscript d represents the number of consecutive secondpolar peptoid monomers within a subunit. In some embodiments, c isbetween 0 and 10 (e.g., c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Inother embodiments, d is between 0 and 10 (e.g., d is 0, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10). In particular embodiments, both c and d are not 0.In some instances, c is between 0 and 5. In other instances, d isbetween 0 and 5.

As non-limiting examples, a subunit of a peptoid polymer of the presentinvention can comprise the sequence HP, PH, HHPP, PPHH, HPHP, PHPH,HPPH, PHHP, HHP, PHH, HPP, PPH, HPPP, PPPH, HHPPHH, PPHHPP, HHHPPP,PPPHHH, HHHP, PHHH, HHHPPPHHH, or PPPHHHPPP. When a is 1 and b is 1, thesubunit can comprise the sequence HP or PH. When a is 2 and b is 2, thesubunit can comprise the sequence HHPP or PPHH. When a is 1 and b is 2,the subunit can comprise the sequence HPP or PPH. When a is 2 and b is1, the subunit can comprise the sequence HHP or PHH. When a is 1 and bis 3, the subunit can comprise the sequence HPPP or PPPH. When a is 3and b is 1, the subunit can comprise the sequence HHHP or PHHH. When ais 3 and b is 3, the subunit can comprise the sequence HHHPPP or PPPHHH.

As further non-limiting examples, when a, b, c, and d are 1, the subunitcan comprise the sequence HPHP or PHPH, although these sequences canalso be represented by the formulas [H₁P₁]₂ and [P₁H₁]₂, respectively,where n is 2. When a is 1, b is 2, c is 1, and d is 0, the subunit cancomprise the sequence HPPH. When a is 2, b is 1, c is 0, and d is 1 thesubunit can comprise the sequence PHHP (i.e., P₁H₂P₁H₀). When a, b, andc are 2 and d is 0, the subunit can comprise the sequence HHPPHH. Whena, b, and d are 2 and c is 0, the subunit can comprise the sequencePPHHPP (i.e., P₂H₂P₂H₀). When a, b, and c are 3 and d is 0, the subunitcan comprise the sequence HHHPPPHHH. When a, b, and d are 3 and c is 0,the subunit can comprise the sequence PPPHHHPPP (i.e., P₃H₃P₃H₀).

In some embodiments, the peptoid polymer further comprises substituentsX and Y such that the peptoid polymer has the sequenceX—[H_(a)P_(b)]_(n)—Y, X—[P_(b)H_(a)]_(n)—Y,X—[H_(a)P_(b)H_(c)P_(d)]_(n)—Y, or X—[P_(b)H_(a)P_(d)H_(c)]_(n)—Y. X andY are independently selected from the group consisting of H, optionallysubstituted C₁₋₈ alkyl, optionally substituted C₁₋₈ acyl, optionallysubstituted C₁₋₈ alkylamino, —OH, —SH, —NH₂, acetyl, carboxy, optionallysubstituted C₁₋₈ hydroxyalkyl, optionally substituted C₁₋₈ alkylamino,optionally substituted C₂₋₈ alkylthio, optionally substituted C₁₋₈carboxyalkyl, and halogen. In some embodiments, X is an acetyl group. Insome embodiments, Y is carboxy. Alternatively, X and Y are takentogether to form a covalent bond. The formation of a covalent bondbetween X and Y results in a circularized form of the peptoid polymer.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, acetyl, carboxy, optionally substituted C₁₋₈hydroxyalkyl, optionally substituted C₁₋₈ alkylamino, optionallysubstituted C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, orhalogen. In other embodiments, X or Y is a secondary amine or a tertiaryamine.

In some embodiments, the peptoid polymer further comprises a sequence Zthat comprises one or more hydrophobic peptoid monomers and/or one ormore polar peptoid monomers. Z can be located before the first subunit,after the last subunit, and/or between one or more subunits. In someinstances, Z comprises one or more hydrophobic peptoid monomers. Inother instances, Z comprises one or more polar peptoid monomers. Inparticular instances, Z comprises one or more hydrophobic peptoidmonomers and one or more polar peptoid monomers. Z can comprise a numberof contiguous hydrophobic peptoid monomers followed by a number ofcontiguous polar peptoid monomers, or vice versa. Alternatively, Z cancomprise a number of contiguous hydrophobic peptoid monomers followed bya number of contiguous polar peptoid monomers, followed by additionalhydrophobic peptoid monomers, and so on. When more than one hydrophobicpeptoid monomer is present in sequence Z, all of the hydrophobic peptoidmonomers can be of the same type, they can each be different, or acombination thereof. Similarly, when more than one polar peptoid monomeris present in sequence Z, all of the polar peptoid monomers can be ofthe same type, they can each be different, or a combination thereof. Insome embodiments, the peptoid polymer comprises more than 1 (e.g., 2, 3,4, 5, or more) instances of a sequence Z. In such cases, all instancesof Z can be the same, they can each be different, or a combinationthereof. In particular embodiments, a sequence Z comprises 1, 2, 3, 4,or more hydrophobic peptoid monomers. In yet other embodiments, asequence Z comprises 1, 2, 3, 4, or more polar peptoid monomers.

In other aspects, provided herein is a peptoid polymer comprising: (a)subunits comprising two first hydrophobic peptoid monomers H and twofirst polar peptoid monomers P, and (b) two second hydrophobic peptoidmonomers located at the C-terminal end of the peptoid polymer, arrangedsuch that the peptoid polymer has the sequence [H₂P₂]_(n)H₂ or[P₂H₂]_(n)H₂, wherein the subscript n, representing the number ofsubunits within the peptoid polymer, is between 1 and 50.

In still other aspects, provided herein is a peptoid polymer comprising:(a) subunits comprising two first hydrophobic peptoid monomers H and twofirst polar peptoid monomers P, and (b) two second polar peptoidmonomers located at the C-terminal end of the peptoid polymer, arrangedsuch that the peptoid polymer has the sequence [H₂P₂]_(n)P₂ or[P₂H₂]_(n)P₂, wherein the subscript n, representing the number ofsubunits within the peptoid polymer, is between 1 and 50.

In varying embodiments, all of the hydrophobic peptoid monomers can bethe same, they can all be different, or a combination thereof.Similarly, all of the polar peptoid monomers can be the same, they canall be different, or a combination thereof.

In some embodiments, the peptoid polymer further comprises substituentsX and Y such that the peptoid polymer has the sequence X—[H₂P₂]_(n)H₂—Y,X—[P₂H₂]_(n)H₂—Y, X—[H₂P₂]_(n)P₂—Y, or X—[P₂H₂]_(n)P₂—Y. In someembodiments, X and Y are independently selected from the groupconsisting of H, optionally substituted C₁₋₈ alkyl, optionallysubstituted C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,—NH₂, acetyl, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,optionally substituted C₁₋₈ alkylamino, optionally substituted C₂₋₈alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and halogen.Alternatively, X and Y are taken together to form a covalent bond. Theformation of a covalent bond between X and Y results in a circularizedform of the peptoid polymer.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, acetyl, carboxy, optionally substituted C₁₋₈hydroxyalkyl, optionally substituted C₁₋₈ alkylamino, optionallysubstituted C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, orhalogen. In other embodiments, X or Y is a secondary amine or a tertiaryamine.

In some embodiments, n is between 1 and 50 (e.g., n is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50). In some instances, n is between 1 and 10(e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n isbetween 1 and 25. In some embodiments, n is between 3 and 25. In someembodiments, n is between 5 and 25. In some embodiments, n is between 8and 50. In some embodiments, n is between 8 and 25. In some embodiments,n is between 8 and 20. In some embodiments, n can be from about 10 toabout 28, from about 12 to about 26, from about 14 to about 24, fromabout 16 to about 22, or from about 18 to about 20. In some embodiments,n can be from about 8 to about 50, from about 8 to about 45, from about8 to about 40, from about 8 to about 35, from about 8 to about 30, fromabout 10 to about 25, from about 10 to about 20, or from about 10 toabout 15.

In some embodiments, the peptoid polymer comprises Compound 62, Compound63, Compound 67, Compound 73, Compound 76, Compound 86, or Compound 87(i.e., when n is 2). In some instances, the peptoid polymer comprisesCompound 76. In other embodiments, the peptoid polymer comprisesCompound 81 (i.e., when n is 1).

In some embodiments, the peptoid polymer comprises one or more of thehydrophobic peptoid monomers selected from the group of monomers setforth in Table 1 above. In some embodiments, the peptoid polymercomprises one or more of the polar peptoid monomers selected from thegroup of monomers set forth in Table 1 above. In some embodiments, thepeptoid polymer comprises one or more of the hydrophobic peptoidmonomers and one or more of the polar peptoid monomers selected from thegroup of monomers set forth in Table 1 above.

In some embodiments, the first and/or second hydrophobic peptoidmonomers are independently selected from the group consisting of

wherein the subscript m is the number of repeat units and is between 1and 10 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In someembodiments, the repeating unit, m, can be between 1 and 2, 1 and 3, 1and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, or 1 and 10.

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more hydrophobic peptoid monomers in thepeptoid polymer have a side chain (e.g., R¹) that comprises anindependently selected alkyl group wherein the alkyl group has 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more carbonatoms. In particular embodiments, the alkyl group has 5 carbon atoms. Insome instances, the 5-carbon alkyl group is a pentyl group. In otherinstances, the 5-carbon alkyl group is a substituted butyl group (e.g.,1-methylbutyl, 2-methylbutyl, 3-methylbutyl, and the like). In yet otherinstances, the 5-carbon alkyl group is a substituted propyl group (e.g.,1-ethylpropyl, 1,2-dimethylpropyl, and the like).

In some embodiments, the first and/or second polar peptoid monomers areindependently selected from the group consisting of

wherein the subscript m is the number of repeat units and is between 1and 10 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In someembodiments, the repeating unit, m, can be between 1 and 2, 1 and 3, 1and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, or 1 and 10.

In some embodiments, the first and/or second polar peptoid monomer has aside chain (e.g., R¹) that comprises a hydroxyl group. In otherembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more polar peptoid monomers in the peptoidpolymer have a side chain (e.g., R¹) that comprises an independentlyselected C₁₋₁₈ hydroxyalkyl group (e.g., an independently selected C₁₋₆hydroxyalkyl group). In some instances, each C₁₋₁₈ hydroxyalkyl group isa C₁₋₆ hydroxyalkyl group. In particular instances, each C₁₋₆hydroxyalkyl group is the same C₁₋₆ hydroxyalkyl group. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more polar peptoid monomers in the peptoidpolymer have a side chain (e.g., R¹) that comprises an independentlyselected hydroxyalkyl group where the length of the alkyl in thehydroxyalkyl group is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or more carbon atoms. In particularembodiments, the hydroxyalkyl group contains 1, 2, 3, 4, 5, 6, 7, or 8hydroxy substitutions.

In some embodiments, none of the polar peptoid monomers comprise a sidechain (e.g., R′) that comprises an optionally substituted C₁₋₁₈hydroxyalkyl group.

In some embodiments, the side chain (e.g., R¹) of a polar peptoidmonomer comprises a (4- to 10-membered heterocycloalkyl)(C₁₋₆ alkylene)group or a (5- to 10-membered heteroaryl)(C₁₋₆ alkylene) group. Thealkylene moiety can be, for example, a straight-chain alkylene moietysuch as methylene, ethylene, n-propylene (i.e., —CH₂CH₂CH₂—), orn-butylene (i.e., —CH₂CH₂CH₂CH₂—). The alkylene linker can also bebranched, as in the case of sec-butylene (i.e., —CH(CH₃)CH₂CH₂—) oriso-butylene (i.e., —CH₂CH(CH₃)CH₂—). In some embodiments, the alkylenemoiety is methylene (i.e., —CH₂—). In some embodiments, the alkylenemoiety is n-propylene. In some embodiments, at least one member of the4-, 5-, 6-, 7-, 8-, 9-, or 10-membered ring is 0. In other embodiments,at least one member of the 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered ringis N.

The heterocycloalkyl moiety can be, but is not limited to, a 4- to8-membered ring, a 4- to 6-membered ring, or a 5- to 6-membered ring.The heterocycloalkyl moiety can be, for example, azetidinyl,pyrrolidinyl, piperidinyl, azepanyl, azocanyl, pyrazolidinyl,imidazolidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl,oxepanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, or morpholinyl. In someembodiments, the heterocycloalkyl moiety is selected frompyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, tetrahydrofuran-2-yl,and tetrahydrofuran-3-yl. In some embodiments, the heterocycloalkylmoiety is pyrrolidin-1-yl. In some embodiments, the heterocycloalkylmoiety is tetrahydrofuran-2-yl. In some embodiments, one or more carbonring members in pyrrolidinyl or tetrahydrofuranyl is substituted withoxo.

In some embodiments, the peptoid polymer comprises

In some instances, the peptoid polymer comprises Compound 63, Compound76, Compound 86, and/or Compound 87. In some embodiments, all of thepolar peptoid monomers are

In other embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 76, Compound 86, orCompound 87.

The heteroaryl moiety can be, but is not limited to, a 5- to 10-memberedring, a 5- to 9-membered ring, or a 5- to 6-membered ring. Theheteroaryl moiety can be, for example, indolyl, quinolinyl,quinoxalinyl, quinazolinyl, phthalazinyl, cinnolinyl, benzofuranyl,pyrrolyl, pyridinyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl,pyridazinyl, thiophenyl, or furanyl. In some embodiments, the heteroarylmoiety is selected from furan-2-yl, furan-3-yl, thiophen-2-yl,thiophen-3-yl, pyrrol-1-yl, pyrrol-2-yl, and pyrrol-3-yl.

In some embodiments, the side chain (e.g., R¹) comprises a(2-oxopyrrolidin-1-yl)(C₁₋₄ alkylene) group. In some embodiments, theside chain (e.g., R¹) comprises a (tetrahydrofuran-2-yl)(C₁₋₄ alkylene)group. In some embodiments, the side chain (e.g., R¹) comprises a(furan-2-yl)(C₁₋₄ alkylene) group.

In some embodiments, the peptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 73.

In some embodiments, the side chain (e.g., R′) of a polar peptoidmonomer comprises a 2- to 20-membered alkoxy group. The side chain(e.g., R′) can comprise, for example, a 2-12 membered alkoxy grouphaving from 1-4 oxygen atoms, or 2-6 membered alkoxy having 1 or 2oxygen atoms. In some embodiments, the side chain (e.g., R′) comprises—CH₂CH₂OR′, wherein R′ is C₁₋₆ alkyl. In some embodiments, the sidechain (e.g., R¹) comprises —CH₂CH₂O(CH₂CH₂O)_(n)R′, wherein R′ is C₁₋₆alkyl and subscript n is 1, 2, or 3.

In some embodiments, the side chain (e.g., R′) comprises a (C₁₋₆alkoxy)(C₁₋₆ alkylene) group. In some embodiments, the side chain (e.g.,R′) comprises an (oligo[ethylene glycol]) or (oligo[propylene glycol])group. In some embodiments, the side chain (e.g., R′) comprises amethoxyethyl group. In some embodiments, the peptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 62.

In some embodiments, the oligo(ethylene glycol) moiety is a2-(2-(2-methoxyethoxy)ethoxy)ethyl moiety. In some embodiments, thepeptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 67.

In yet other aspects, provided herein is a peptoid polymer comprisingone or more hydrophobic peptoid monomers and one or more polar peptoidmonomers. In some embodiments, none of the polar peptoid monomerscomprise a side chain (e.g., R¹) that comprises an optionallysubstituted C₁₋₁₈ hydroxyalkyl group. In some embodiments, the sidechain (e.g., R¹) of a polar peptoid monomer comprises a (4- to10-membered heterocycloalkyl)(C₁₋₆ alkylene) group. In otherembodiments, the side chain (e.g., R¹) of a polar peptoid monomercomprises a (5- to 10-membered heteroaryl)(C₁₋₆ alkylene) group. In someembodiments, the side chain (e.g., R¹) of a polar peptoid monomercomprises a 2- to 20-membered alkoxy group. The side chain (e.g., R¹)can comprise, for example, 2-12 membered alkoxy having from 1-4 oxygenatoms, or 2-6 membered alkoxy having 1 or 2 oxygen atoms. In someembodiments, the side chain (e.g., R¹) comprises —CH₂CH₂OR′, wherein R′is C₁₋₆ alkyl. In some embodiments, the side chain (e.g., R′) comprises—CH₂CH₂O(CH₂CH₂O)_(n)R′, wherein R′ is C₁₋₆ alkyl and subscript n is 1,2, or 3. In some embodiments, the side chain (e.g., R′) of a polarpeptoid monomer comprises a (C₁₋₆ alkoxy)(C₁₋₆ alkylene) group. In someembodiments, the side chain (e.g., R′) of a polar peptoid monomercomprises an (oligo[ethylene glycol]) or (oligo[propylene glycol])group.

The alkylene moiety can be, for example, a straight-chain alkylenemoiety such as methylene, ethylene, n-propylene (i.e., —CH₂CH₂CH₂—), orn-butylene (i.e., —CH₂CH₂CH₂CH₂—). The alkylene linker can also bebranched, as in the case of sec-butylene (i.e., —CH(CH₃)CH₂CH₂—) oriso-butylene (i.e., —CH₂CH(CH₃)CH₂—). In some embodiments, the alkylenemoiety is methylene (i.e., —CH₂—). In some embodiments, the alkylenemoiety is n-propylene. In some embodiments, at least one member of the4-, 5-, 6-, 7-, 8-, 9-, or 10-membered ring is 0. In other embodiments,at least one member of the 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered ringis N.

The heterocycloalkyl moiety can be, but is not limited to, a 4- to8-membered ring, a 4- to 6-membered ring, or a 5- to 6-membered ring.The heterocycloalkyl moiety can be, for example, azetidinyl,pyrrolidinyl, piperidinyl, azepanyl, azocanyl, pyrazolidinyl,imidazolidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl,oxepanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, or morpholinyl. In someembodiments, the heterocycloalkyl moiety is selected frompyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, tetrahydrofuran-2-yl,and tetrahydrofuran-3-yl. In some embodiments, the heterocycloalkylmoiety is pyrrolidin-1-yl. In some embodiments, the heterocycloalkylmoiety is tetrahydrofuran-2-yl. In some embodiments, one or more carbonring members in pyrrolidinyl or tetrahydrofuranyl is substituted withoxo.

In some embodiments, the peptoid polymer comprises

In some instances, the peptoid polymer comprises Compound 63, Compound76, Compound 86, and/or Compound 87. In some embodiments, all of thepolar peptoid monomers are

In other embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 76, Compound 86, orCompound 87.

The heteroaryl moiety can be, but is not limited to, a 5- to 10-memberedring, a 5- to 9-membered ring, or a 5- to 6-membered ring. Theheteroaryl moiety can be, for example, indolyl, quinolinyl,quinoxalinyl, quinazolinyl, phthalazinyl, cinnolinyl, benzofuranyl,pyrrolyl, pyridinyl, imidazolyl, pyrazolyl, pyrazinyl, pyrimidinyl,pyridazinyl, thiophenyl, or furanyl. In some embodiments, the heteroarylmoiety is selected from furan-2-yl, furan-3-yl, thiophen-2-yl,thiophen-3-yl, pyrrol-1-yl, pyrrol-2-yl, and pyrrol-3-yl.

In some embodiments, the side chain (e.g., R¹) comprises a(2-oxopyrrolidin-1-yl)(C₁₋₄ alkylene) group. In some embodiments, theside chain (e.g., R¹) comprises a (tetrahydrofuran-2-yl)(C₁₋₄ alkylene)group. In some embodiments, the side chain (e.g., R¹) comprises a(furan-2-yl)(C₁₋₄ alkylene).

In some embodiments, the peptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 73.

In some embodiments, the side chain (e.g., R¹) comprises a methoxyethylgroup. In some embodiments, the peptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 62.

In some embodiments, the oligo(ethylene glycol) moiety is a2-(2-(2-methoxyethoxy)ethoxy)ethyl moiety. In some embodiments, thepeptoid polymer comprises

In particular embodiments, all of the polar peptoid monomers are

In some instances, the peptoid polymer is Compound 67.

In some embodiments, each of the one or more hydrophobic peptoidmonomers are independently selected from the group consisting of

wherein the subscript m is the number of repeat units and is between 1and 10.

In some embodiments, each of the one or more polar peptoid monomers areindependently selected from the group consisting of

In some embodiments, the peptoid polymer further comprises substituentsX and Y located at the N-terminal and C-terminal ends of the peptoidpolymer, respectively. In some embodiments, X and Y are independentlyselected from the group consisting of H, optionally substituted C₁₋₈alkyl, optionally substituted C₁₋₈ acyl, optionally substituted C₁₋₈alkylamino, —OH, —SH, —NH₂, acetyl, carboxy, optionally substituted C₁₋₈hydroxyalkyl, optionally substituted C₁₋₈ alkylamino, optionallysubstituted C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl,and halogen. Alternatively, X and Y are taken together to form acovalent bond. The formation of a covalent bond between X and Y resultsin a circularized form of the peptoid polymer.

In some embodiments, X and Y are H, optionally substituted C₁₋₈alkylamino, —OH, —SH, acetyl, carboxy, optionally substituted C₁₋₈hydroxyalkyl, optionally substituted C₁₋₈ alkylamino, optionallysubstituted C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, orhalogen. In other embodiments, X or Y is a secondary amine or a tertiaryamine.

Whenever any peptoid monomer described herein does not indicatestereochemistry, any stereoisomer may be used. In some embodiments, amixture of stereoisomers are chosen. Non-limiting examples ofstereoisomers of hydrophobic peptoid monomers and polar peptoid monomersas well as exemplary ratios of R to S stereoisomers of the peptoidmonomers in the peptoid polymers are described above.

In some embodiments, between about 1 percent and about 99 percent (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99 percent) of the peptoid monomers inthe peptoid polymer are hydrophobic peptoid monomers. In otherembodiments, between about 1 percent and about 99 percent (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99 percent) of the peptoid monomers in thepeptoid polymer are polar peptoid monomers.

In some embodiments, about 5 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 95 percent of the peptoidmonomers are polar.

In some embodiments, about 10 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 90 percent of the peptoidmonomers are polar.

In some embodiments, about 15 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 85 percent of the peptoidmonomers are polar.

In some embodiments, about 20 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 80 percent of the peptoidmonomers are polar.

In some embodiments, about 25 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 75 percent of the peptoidmonomers are polar.

In some embodiments, about 30 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 70 percent of the peptoidmonomers are polar.

In some embodiments, about 35 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 65 percent of the peptoidmonomers are polar.

In some embodiments, about 40 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 60 percent of the peptoidmonomers are polar.

In some embodiments, about 45 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 55 percent of the peptoidmonomers are polar.

In some embodiments, about 50 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 50 percent of the peptoidmonomers are polar.

In some embodiments, about 55 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 45 percent of the peptoidmonomers are polar.

In some embodiments, about 60 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 40 percent of the peptoidmonomers are polar.

In some embodiments, about 65 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 35 percent of the peptoidmonomers are polar.

In some embodiments, about 70 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 30 percent of the peptoidmonomers are polar.

In some embodiments, about 75 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 25 percent of the peptoidmonomers are polar.

In some embodiments, about 80 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 20 percent of the peptoidmonomers are polar.

In some embodiments, about 85 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 15 percent of the peptoidmonomers are polar.

In some embodiments, about 90 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 10 percent of the peptoidmonomers are polar.

In some embodiments, about 95 percent of the peptoid monomers in thepeptoid polymer are hydrophobic and about 5 percent of the peptoidmonomers are polar.

In particular embodiments, the peptoid polymer comprises about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95percent hydrophobic peptoid monomers by mass. In other embodiments, thepeptoid polymer comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, or 95 percent polar peptoid monomers bymass.

In some embodiments, the peptoid polymer described herein forms ahelical structure. In some embodiments, the helical structure adopts astructure analogous to a polyproline helix. In certain instances, thepeptoid polymer forms a polyproline I helix. In certain other instances,the peptoid polymer forms a polyproline II helix. In some embodiments, ahelical structure is adopted when the peptoid polymer comprises at leastone N-Aryl side chain. In some embodiments, the N-Aryl side chain is aNep monomer.

In some embodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about 0° C. to about −20° C. In otherembodiments, the peptoid polymer reduces or inhibits ice crystalformation at a temperature within about −10° C. to about −20° C. Incertain embodiments, the peptoid polymer reduces or inhibits ice crystalformation at about −5° C., about −10° C., about −15° C., or about −20°C.

In some embodiments, the concentration of the peptoid polymer (e.g.,present in a composition, formulation, or product such as acryoprotectant solution) is between about 100 nM and about 1 M. Incertain embodiments, the concentration of the peptoid polymer (e.g.,present in a composition, formulation, or product such as acryoprotectant solution) is between about 100 nM and about 250 nM,between about 250 nM and about 500 nM, between about 500 nM and about750 nM, between about 750 nM and about 1 μM, between about 1 μM andabout 5 μM, between about 5 μM and about 25 μM, between about 25 μM andabout 50 μM, between about 50 μM and about 100 μM, between about 100 μMand about 250 μM, between about 250 μM and about 500 μM, between about500 μM and about 750 μM, between about 750 μM and about 1 mM, betweenabout 1 mM and about 10 mM, between about 10 mM and about 50 mM, betweenabout 50 mM and about 100 mM, between about 100 mM and about 250 mM,between about 250 mM and about 500 mM, between about 500 mM and about750 mM, or between about 750 mM and about 1 M. In some embodiments, theconcentration of the peptoid polymer (e.g., present in a composition,formulation, or product such as a cryoprotectant solution) is betweenabout 100 nM and about 100 mM. In other embodiments, the concentrationof the peptoid polymer (e.g., present in a composition, formulation, orproduct such as a cryoprotectant solution) is about 100 nM, about 500nM, about 1 μM, about 10 μM, about 100 μM, about 500 μM, about 1 mM,about 10 mM, about 100 mM, about 500 mM, or about 1 M. In particularembodiments, the concentration of the peptoid polymer is between about 1and 100 mM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, or 100 mM).

B. Peptoid-Peptide Hybrids

In another aspect, the invention provides a peptoid-peptide hybrid. Insome embodiments, the peptoid-peptide hybrid comprises a peptoid polymerdescribed herein and one or more amino acids. The amino acids can benaturally-occurring amino acids or variants thereof. In someembodiments, the peptoid-peptide hybrid comprises between about 1 and 10amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).In other embodiments, the peptoid-peptide hybrid comprises between about10 and 100 amino acids (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids). In someembodiments, the peptoid-peptide hybrid comprises more than about 100amino acids. In other embodiments, the peptoid-peptide hybrid comprisesbetween 2 and 50 peptoid monomers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 44, 45, 46, 47,48, 49, or 50 peptoid monomers) and at least between about 1 and 100amino acids (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacids).

The amino acids can be located at any position within the polymer,including at the N- and C-terminal ends and/or in between any of thepeptoid monomers or subunits. In instances where the peptoid-peptidehybrid comprises two or more amino acids, the amino acids may all becontiguous, or only a portion of them may be contiguous. Alternatively,all of the amino acids may be separated by one or more peptoid monomersor subunits.

In some embodiments, the amino acids are D-amino acids. In otherembodiments, the amino acids are L-amino acids. In some otherembodiments, the peptoid-peptide hybrid comprises a combination of D-and L-amino acids. In some embodiments, the one or more amino acids areselected from the group consisting of alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, arginine,lysine, leucine, methionine, asparagine, proline, glutamine, serine,threonine, valine, tryptophan, tyrosine, and a combination thereof. Insome instances, the one or more amino acids are selected from the groupconsisting of isoleucine, threonine, alanine, and a combination thereof.

In some embodiments, one or more Nsb peptoid monomers in a peptoidpolymer are replaced with one or more isoleucine amino acid residues tocreate a peptoid-peptide hybrid. The one or more isoleucine amino acidscan be D-amino acids, L-amino acids, or a combination thereof. In otherembodiments, one or more Nhp peptoid monomers in a peptoid polymer arereplaced with one or more threonine amino acid residues to create apeptoid-peptide hybrid. The one or more threonine amino acids can beD-amino acids, L-amino acids, or a combination thereof. In some otherembodiments, one or more Nme peptoid monomers in a peptoid polymer arereplaced with one or more alanine amino acid residues to create apeptoid-peptide hybrid. The one or more alanine amino acids can beD-amino acids, L-amino acids, or a combination thereof.

In some embodiments, the peptoid-peptide hybrid comprises the sequence:

Nep-Nep-Xaa-Xaa-Xaa-Xaa-Nep-Nep-Nep-Nep-Nme-Nme;wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orSer amino acid residues.

In other embodiments, the peptoid-peptide hybrid comprises the sequence:

Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nhp-Nhp-Nsb-Xaa-Nme- Nme-Xaa-Nme-Nme-Nme;wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orIle amino acid residues.

In yet other embodiments, the peptoid-peptide hybrid comprises thesequence:

Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nme-Nme-Nme-Xaa-Xaa;wherein the Xaa amino acid residues are independently selected aminoacids such as D-amino acids, L-amino acids, or a combination thereof. Asa non-limiting example, all instances of Xaa are Arg, Ala, Val, and/orLeu amino acid residues.

In some embodiments, the peptoid-peptide hybrid comprises the sequence:

Arg-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb;wherein the Arg amino acid residue is a D-amino acid or an L-amino acid.In some embodiments, the peptoid-peptide hybrid comprises the structureset forth in Table 11.

TABLE 11

Compound 58

C. Methods of Synthesis

In another aspect, the invention herein provides a method ofsynthesizing a peptoid polymer or a peptoid-peptide hybrid. The peptoidpolymers and peptoid-peptide hybrids of the invention can be preparedfrom readily available starting materials using the general methods andprocedures described herein. It will be appreciated that where typicalor preferred process conditions (i.e., reaction temperatures, times,mole ratios of reactants, solvents, pressures, etc.) are given, otherprocess conditions can also be used unless otherwise stated. Optimumreaction conditions may vary with the particular reactants or solventused, but such conditions can be determined by one skilled in the art byroutine optimization procedures.

The peptoid polymers and peptoid-peptide hybrids of the invention may beprepared from known or commercially available starting materials andreagents by one skilled in the art of organic synthesis. Solvents andreagents are purchased from commercial sources and used without furtherpurification.

In some embodiments, the submonomer approach (FIG. 1 ) is used forpeptoid synthesis, where each N-substituted glycine monomer is assembledfrom two readily available “submonomers.” The synthesis of oligomericpeptoids is based on the robust chemistry of standard solid-phasemethods, analogous to peptide synthesis. Each cycle of monomer additionconsists of two steps, an acylation step and a nucleophilic displacementstep. In some embodiments, solid-phase assembly eliminates the need forN-protected monomers because there are no reactive side chainfunctionalities that need to be protected. One of skill in the art willrecognize there are many solid-phase synthesis methods, includingautomated, robotic synthesizers. In some embodiments, the synthesizerused is the Symphony® X Multiplex Peptide Synthesizer made by ProteinTechnologies, Inc. In some embodiments, the synthesizer used is theOverture Peptide Synthesizer made by Protein Technologies, Inc. In otherembodiments, the peptoids are synthesized manually using traditionalorganic chemistry methods known in the art. By providing the appropriateamino acids in place of peptoid monomers at the appropriate times duringsynthesis, the same techniques or techniques similar to those describedabove can be applied to the synthesis of peptoid-peptide oligomers.

As a non-limiting example, peptoid polymers can be synthesized on 100 mgof Rink amide resin (NovaBiochem; 0.49 mmol/g). Rink amide resin (100mg) can be washed twice in 1.5 mL of DCM, followed by swelling in 1.5 mLof DMF. The swelling step can be performed twice. The Fmoc protectinggroup can be removed from the resin by addition of 20% piperidine/DMF.The mixture can be agitated for 10 minutes, drained, and the piperidinetreatment repeated, followed by extensive washes with DMF (five timeswith 1.5 mL). The first monomer can be added manually by reacting 37 mgof bromoacetic acid (0.27 mmol; Sigma-Aldrich) and 189 μL of DIEA (1.08mmol; Chem Impex International) in 2 mL of DCM on a shaker platform for30 minutes at room temperature, followed by extensive washes with DCM(five times with 2 mL) and DMF (five times with 2 mL). Bromoacylatedresin can be incubated with 2 mL of 1 M amine submonomer in DMF on ashaker platform for 30 minutes at room temperature, followed byextensive washes with DMF (five times with 2 mL). After initial manualloading of bromoacetic acid, the first submonomer displacement step andall subsequent bromo acetylation and amine displacement steps can beperformed by a robotic synthesizer until the desired oligomer length isobtained. The automated bromoacetylation step can be performed by adding1660 μL of 1.2 M bromoacetic acid in DMF and 400 μL of DIC (Chem ImpexInternational). The mixture can be agitated for 20 min, drained, andwashed with DMF (three times with 2 mL). Next, 2 mL of a 1 M solution ofsubmonomer (2 mmol) in DMF can be added to introduce the side chain bynucleophilic displacement of bromide. The mixture can be agitated for 20min, drained, washed with DMF (three times with 2 mL) and washed withDCM (three times with 2 mL). The peptoid-resin can be cleaved in 2 mL of20% HFIP (Alfa Aesar) in DCM (v/v) at room temperature. The cleavage canbe conducted in a glass tube with constant agitation for 30 minutes.HFIP/DCM can be evaporated under a stream of nitrogen gas. The finalproduct can be dissolved in 5 mL of 50% ACN in HPLC grade H₂O andfiltered with a 0.5 pm stainless steel fritted syringe tip filter(Upchurch Scientific). Peptoid oligomers can be analyzed on a C18reversed-phase analytical HPLC column at room temperature (PeekeScientific, 5 μm, 120 Å, 2.0×50 mm) using a Beckman Coulter System Goldinstrument. A linear gradient of 5-95% acetonitrile/water (0.1% TFA,Acros Organics) over 20 min can be used with a flow rate of 0.7 mL/min.In order to remove any traces of HFIP in the sample solution, linearprecursors dissolved in 50% ACN/H₂O can be freeze-dried overnight.

Peptoid polymers and peptoid-peptide hybrids can be analyzed byelectrospray ionization (ESI) mass spectrometry. Generally, 0.5-2 mL of1-5 μM of peptoid polymer or peptoid-peptide hybrid to be analyzed isprepared in a 50% deionized H₂O/50% HPLC grade ACN with 1% of an organicacid such as trifluoroacetic acid. Prepared samples are ionized bybombardment with electrons causing the molecules to break into chargedfragments. The ions are then separated according to their mass-to-chargeratio by accelerating the fragments and exposing them to an electricalor magnetic field. The ions are detected by a mechanism capable ofdetecting charged particles, such as an electron multiplier. Peptoidsand peptoid-peptide hybrids are identified by correlating masses to theidentified masses or through a characteristic fragmentation pattern.

D. Methods of Use

In some aspects, the present invention provides a cryoprotectantsolution. In some embodiments, the cryoprotectant solution comprises apeptoid polymer described herein, a peptoid-peptide hybrid describedherein, or a combination thereof. In other embodiments, thecryoprotectant solution further comprises a compound selected from thegroup consisting of an ionic species, a penetrating cryoprotectant, anon-penetrating cryoprotectant, an antioxidant, a cell membranestabilizing compound, an aquaporin or other channel forming compound, analcohol, a sugar, a sugar derivative, a nonionic surfactant, a protein,dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polypropyleneglycol (PPG), Ficoll®, polyvinylpyrrolidone, polyvinyl alcohol,hyaluronan, formamide, a natural or synthetic hydrogel, and acombination thereof. In particular embodiments, the penetratingcryoprotectant penetrates the cell membrane and reduces theintracellular water concentration, thereby reducing the amount of iceformed at any temperature. In other particular embodiments, thenon-penetrating cryoprotectant induces changes in colloidal osmoticpressure and modifies cell membrane associations with extracellularwater by induced ionic interaction.

In some instances, the cryoprotectant solution further comprises analcohol that is selected from the group consisting of propylene glycol,ethylene glycol, glycerol, methanol, butylene glycol, adonitol, ethanol,trimethylene glycol, diethylene glycol, polyethylene oxide, erythritol,sorbitol, xythyritol, polypropylene glycol, 2-methyl-2,4-pentanediol(MPD), mannitol, inositol, dithioritol, 1,2-propanediol, and acombination thereof.

In other instances, the cryoprotectant solution further comprises asugar that is selected from the group consisting of a monosaccharide, adisaccharide, a polysaccharide, and a combination thereof. In particularinstances, the sugar is selected from the group consisting of glucose,3-O-Methyl-D-glucopyranose, galactose, arabinose, fructose, xylose,mannose, sucrose, trehalose, lactose, maltose, raffinose, dextran, and acombination thereof.

In other instances, the cryoprotectant solution further comprises PEG,PPG, or a plurality of different PEG or PPG compounds. In some otherinstances, at least one of the PEG or PPG compounds has an averagemolecular weight less than about 3,000 g/mol (e.g., less than about3,000, 2,500, 2,000, 1,500, 1,000, 950, 900, 850, 800, 750, 700, 650,600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 g/mol). Inparticular instances, at least one of the PEG or PPG compounds has anaverage molecular weight between about 200 and 400 g/mol (e.g., about200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, or 400 g/mol). In some instances, thecryoprotectant solution comprises PEG or a plurality of PEG compoundsselected from the group consisting of PEG 200, PEG 300, PEG 400, and acombination thereof.

In other instances, the cryoprotectant solution further comprises aprotein that is selected from the group consisting of egg albumin,bovine serum albumin, human serum albumin, gelatin, and a combinationthereof. In still other instances, the cryoprotectant solution furthercomprises a natural or synthetic hydrogel, wherein the natural orsynthetic hydrogel comprises chitosan, hyaluronic acid, or a combinationthereof.

Non-limiting examples of various properties of the cryoprotectantsolution such as effective concentration, viscosity, water solubility,and/or membrane permeability can be assessed using a model cell ortissue including, but not limited to, genitourinary cells (e.g., corpuscavernosum cells such as smooth muscle corpus cavernosum cells and/orepithelial corpus cavernosum cells), stem cells, liver tissue orhepatocytes, kidney, intestine, heart, pancreas, bone marrow, organoids,and other biological tissues for cryopreservation.

In some embodiments, the cryoprotectant solution reduces or inhibits icecrystal formation at a temperature within about 0° C. to about −20° C.In other embodiments, the cryoprotectant solution reduces or inhibitsice crystal formation at a temperature within about −10° C. to about−20° C. In certain embodiments, the cryoprotectant solution reduces orinhibits ice crystal formation at about −5° C., about −10° C., about−15° C., or about −20° C.

In some embodiments, the concentration of the peptoid polymer and/orpeptoid-peptide hybrid in the cryoprotectant solution is between about100 nM and about 1 M. In some embodiments, the concentration of peptoidpolymer and/or peptoid-peptide hybrid in the cryoprotectant solution isbetween about 100 nM and about 250 nM, between about 250 nM and about500 nM, between about 500 nM and about 750 nM, between about 750 nM andabout 1 μM, between about 1 μM and about 5 μM, between about 5 μM andabout 25 μM, between about 25 μM and about 50 μM, between about 50 μMand about 100 μM, between about 100 μM and about 250 μM, between about250 μM and about 500 μM, between about 500 μM and about 750 μM, betweenabout 750 μM and about 1 mM, between about 1 mM and about 10 mM, betweenabout 10 mM and about 50 mM, between about 50 mM and about 100 mM,between about 100 mM and about 250 mM, between about 250 mM and about500 mM, between about 500 mM and about 750 mM, or between about 750 mMand about 1 M. In some embodiments, the concentration of the peptoidpolymer and/or peptoid-peptide hybrid in the cryoprotectant solution isbetween about 100 nM and about 100 mM. In other embodiments, theconcentration of the peptoid polymer and/or peptoid-peptide hybrid inthe cryoprotectant solution is about 100 nM, about 500 nM, about 1 μM,about 10 μM, about 100 μM, about 500 μM, about 1 mM, about 10 mM, about100 mM, about 500 mM, or about 1 M. In particular embodiments, theconcentration of the peptoid polymer and/or peptoid-peptide hybrid inthe cryoprotectant solution is between about 1 and 100 mM (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100mM).

In other aspects, provided herein is a method for preserving abiological sample. In particular embodiments, the biological samplepossesses cellular composition. In some embodiments, the biologicalsample is a cell. In some embodiments, the biological sample comprisesprimary cells. In other embodiments, the biological sample is a tissueor an organ. In particular embodiments, the biological sample comprisesone or more cells, tissues, organs, or a combination thereof. In someembodiments, the method comprises contacting the biological sample witha peptoid polymer described herein, a peptoid-peptide hybrid describedherein, a cryoprotectant solution described herein, or a combinationthereof. In some instances, when a combination of compositions orsolutions is used, contacting the biological sample with thecompositions or solutions can be accomplished in multiple steps. As anon-limiting example, a biological sample can first be contacted with apeptoid polymer described herein, and then at a later point thebiological sample can be contacted with a cryoprotectant solutiondescribed herein.

In particular instances, the tissue is a bioengineered tissue. In someinstances, the biological sample is selected from the group consistingof heart, liver, lung, kidney, pancreas, intestine, thymus, cornea,nerve cells, blood platelets, sperm cells, oocytes, embryonic cells,stem cells, bone cells, and a combination thereof. In other instances,the biological sample comprises a population of cells (e.g., primarycells) selected from the group consisting of heart cells, liver cells,lung cells, kidney cells, pancreatic cells, gastric cells, intestinalcells, muscle cells, skin cells, neural cells, blood cells, immunecells, fibroblasts, genitourinary cells, bone cells, stem cells, spermcells, oocytes, embryonic cells, epithelial cells, endothelial cells,and a combination thereof.

Cryoprotection of biological samples is useful for any number ofpurposes. Non-limiting examples include organoid preservation, stem cellpreservation (e.g., hematopoietic stem cells, embryonic stem (ES) cells,pluripotent stem cells (PSCs), and induced pluripotent stem cells(iPSCs)), preservation of adult cells and cell lines (e.g., lymphocytes,granulocytes, immune system cells, bone cells), preservation of embryos,sperm, and oocytes, tissue preservation, and organ preservation.Preservation of tissues, organs, and other biological samples andstructures is especially useful, for example, in the field of organtransplantation. Other useful applications of the present invention tobiological sample cryoprotection will readily be known to one of skillin the art.

In yet other aspects, provided herein is a method for preserving one ormore biological macromolecules. Said biological macromolecules can benaturally or unnaturally occurring. Non-limiting examples of biologicalmacromolecules suitable for cryoprotection by the methods of the presentinvention include nucleic acids (e.g., DNA, RNA), amino acids, proteins,peptides, lipids, and composite structures (e.g., liposomes). In someembodiments, the method comprises contacting the biologicalmacromolecule with a peptoid polymer described herein, a peptoid-peptidehybrid described herein, a cryoprotectant solution described herein, ora combination thereof. In some instances, the biological macromoleculeis an isolated protein. In particular instances, the isolated protein isa protease protein. In some instances, when a combination ofcompositions or solutions is used, contacting the one or more biologicalmacromolecules with the compositions or solutions can be accomplished inmultiple steps. As a non-limiting example, the one or more biologicalmacromolecules can first be contacted with a peptoid polymer describedherein, and then at a later point the biological sample can be contactedwith a cryoprotectant solution described herein.

Cryoprotection of biological macromolecules is useful for any number ofpurposes. Non-limiting examples of such purposes include thepreservation of DNA (e.g., genomic DNA) and RNA samples, thepreservation of stem cell growth factors, and the preservation ofantibodies. Other useful purposes and applications of the presentinvention will be readily known by one of skill in the art.

Biological samples and macromolecules suitable for cryoprotectionaccording to the present invention can come from any biological kingdom(e.g., Animalia (including but not limited to humans and livestockanimals), Plantae, Fungi (including but not limited to mushrooms),Protista, Archaea/Archaeabacteria, and Bacteria/Eubacteria).

E. Cryopreservation Protocols

The methods described herein are useful for cryopreservation duringsupercooling to high sub-zero temperatures (e.g., 0° C. to −20° C.). Inthe field of organ transplantation, organs are typically cooled on ice(e.g., to 0° C. to 4° C.), which limits the transplantation window toabout ten hours. By using ex vivo machine perfusion with cryoprotectantscontaining standard small molecule CPAs, it has been possible topreserve organs for up to 96 hours at a temperature of −6° C. While itis desirable to further reduce the cryopreservation temperature below−6° C., which would extend the possible cryopreservation time, it hasnot been possible to do so because the high concentrations of standardCPAs necessary to further reduce the temperature result in irreversibleorgan damage owing to CPA-related toxicity. For more information, see,e.g., Uygun K, et. al. Nat. Protoc. 10(3):484-94 (2015). Employing exvivo perfusion methods or otherwise contacting biological samples (e.g.,organs and tissues) or macromolecules with peptoid polymers,peptoid-peptide hybrids, and/or cryoprotectant solutions describedherein is useful for supercooling to high sub-zero temperatures,allowing cryopreservation for longer periods of time and at lowertemperatures than is currently feasible. Other suitable applications ofthe present invention to high sub-zero temperature supercooling willreadily be known to one of skill in the art.

One of skill in the art will readily appreciate that the concentrationsand compositions of the peptoid polymers, peptoid-peptide hybrids, andcryoprotectant solutions described herein can be modified depending onthe particular biological sample and/or macromolecule beingcryopreserved and the particular cryopreservation protocol beingemployed.

F. Methods of Screening

In a related aspect, provided herein are methods for screening peptoidpolymers, peptoid-peptide hybrids, and/or cryoprotectant solutions foractivity.

In one embodiment, the peptoid polymer, peptoid-peptide hybrid, and/orcryoprotectant solution is screened for lowering the freezing point ofwater using a polarized light microscope to detect ice crystalformation. Polarized light microscopy is an optical microscopy techniquethat uses polarized light as the light source. Image contrast arisesfrom the interaction of plane-polarized light with a birefringent (ordoubly-refracting) species to produce two individual wave componentsthat are each polarized in mutually perpendicular planes. The velocitiesof these components, which are termed the ordinary and the extraordinarywavefronts, are different and vary with the propagation directionthrough the specimen. After exiting the specimen, the light componentsbecome out of phase, but are recombined with constructive anddestructive interference when they pass through the analyzer. Thisinterference creates a detectable contrast in the sample. Ice crystalformation is easily detected using this technique because ice crystalsare birefringent species. In a standard experiment, samples comprisingthe peptoid polymer, peptoid-peptide hybrid, and/or cryoprotectantsolution are cooled to a desired temperature for a desired amount oftime. One or more samples, while at the desired temperature, are placedunder the polarized light microscope and visually inspected forformation of ice crystals.

In one embodiment, the peptoid polymer, peptoid-peptide hybrid, and/orcryoprotectant solution is screened for lowering the freezing point ofan aqueous solution using differential scanning calorimetry toquantitate thermal hysteresis activity. Differential scanningcalorimetry is a thermoanalytical technique in which the difference inthe amount of heat required to increase the temperature of a sample andreference is measured as a function of temperature. When a physicaltransformation such as phase transition occurs, more or less heat willneed to flow to the sample than the reference to maintain both at thesame temperature. The difference in temperature between the phasetransition of the reference and the sample reports on the sample'sability to reduce or inhibit ice crystal formation at sub 0° C.temperatures. In a standard experiment, a sample comprising the peptoidpolymer, peptoid-peptide hybrid, and/or cryoprotectant solution iscompared to a reference that lacks the peptoid polymer, peptoid-peptidehybrid, and/or cryoprotectant solution.

G. Cell Viability Assays to Test for Activity

In a related aspect, provided herein are cell viability assays to testfor the ability of the peptoid polymer, peptoid-peptide hybrid, and/orcryoprotectant solution to maintain cell viability (e.g., after storage)at reduced temperatures.

In some embodiments, cell viability is tested using the alamarBlue® CellViability Assay Protocol provided by Thermo Fisher Scientific, Inc.Briefly, alamarBlue® is the trade name of resazurin(7-Hydroxy-3H-phenoxazin-3-one 10-oxide) which is a non-toxic cellpermeable compound that is blue in color and virtually non-fluorescent.Upon entering cells, resazurin is reduced to resorufin, a compound thatis red in color and highly fluorescent. Viable cells continuouslyconvert resazurin to resorufin, increasing the overall fluorescence andcolor of the media surrounding cells. Non-viable cells do not convertresazurin to resorufin, thus the overall fluorescence and color of themedia surrounding the cells is an indication of the relative amount ofviable cells in the sample. In a standard experiment, cells and thepeptoid polymer, peptoid-peptide hybrid, and/or cryoprotectant solutionare mixed in any suitable container. The mixture is then cooled to thedesired sub 0° C. temperature and held for the desired amount of time.Cells are then returned to ambient temperatures and the almarBlue®reagent is added, incubated, and measured following the Thermo Fisherprotocol. Typically, direct readout of cell viability is determined bymeasuring the relative fluorescence of the samples at the wavelengthsλ_(Ex)˜560 nm/λ_(Em)˜590 nm.

In some embodiments, cell viability is tested using the LIVE/DEAD®Viability/Cytotoxicity Kit, for mammal cells provided by Thermo FisherScientific, Inc. This kit uses two indicator molecules: calcein AM andEthidium homodoimer-1 (EthD-1). Live cells are distinguished by thepresence of ubiquitous intracellular esterase activity, determined bythe enzymatic conversion of the virtually nonfluorescent cell-permeantcalcein AM to the intensely fluorescent calcein. The polyanionic dyecalcein is well retained within live cells, producing an intense uniformgreen fluorescence in live cells (λ_(Ex)˜495 nm/λ_(Ex)˜515 nm).Conversely, EthD-1 enters cells with damaged membranes and undergoes a40-fold enhancement of fluorescence upon binding to nucleic acids,thereby producing a bright red fluorescence in dead cells (λ_(Ex)−495nm/λ_(Em)˜635 nm). Notably, EthD-1 is excluded by the intact plasmamembrane of live cells, so the determination of live and dead cells iseasily distinguishable. Calcein and EthD-1 can be viewed simultaneouslywith a conventional fluorescein longpass filter. Alternatively, thefluorescence from these dyes may also be observed separately; calceincan be viewed with a standard fluorescein bandpass filter, and EthD-1can be viewed with filters for propidium iodide or Texas Red® dye. In astandard experiment, cells and the peptoid polymer, peptoid-peptidehybrid, and/or cryoprotectant solution are mixed in any suitablecontainer. The mixture is then cooled to the desired sub 0° C.temperature, held at that temperature for the desired amount of time,and then returned to ambient temperatures. Subsequent steps involvingthe addition of the calcein AM and EthD-1 reagents and measuring theassay results are performed as described in the Thermo Fisher protocol.Typically, direct readout of cell viability is determined by measuringthe relative fluorescence at the above indicated wavelengths for bothreagents.

In some embodiments, cell viability is tested using the MTT assay. TheMTT assay is a colorimetric cell viability and proliferation assay thatrelies upon the reduction of yellow tetrazolium MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to theinsoluble formazan, which has a purple color. Tetrazolium dye reductionis dependent on NAD(P)H-dependent oxidoreductase enzymes, primarilylocated in the cytosolic compartment of metabolically active cells. TheMTT assay is available, for example, from ATCC (www.atcc.org) orSigma-Aldrich (www.sigmaaldrich.com). In a standard experiment, cellsand the peptoid polymer, peptoid-peptide hybrid, and/or cryoprotectantsolution are mixed in any suitable container. The mixture is then cooledto the desired sub 0° C. temperature and held for the desired amount oftime. Cells are then returned to ambient temperatures and the MTTreagent is added, incubated, and measured following the ATCC orSigma-Aldrich protocol. Typically, absorbance of converted dye ismeasured at a wavelength of 570 nm with background subtraction at630-690 nm.

IV. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1. Peptoid-Mediated Inhibition of Ice Crystal Formation

This example illustrates the ice crystal inhibition properties ofN-substituted peptoid polymers and peptoid-peptide hybrids at sub 0° C.temperatures.

Capillary Tube Assays

In this experiment, four water-based samples were prepared in capillarytubes containing MilliQ purified water. One sample contained only water,and another sample contained 160 mM ethylene glycol (EG). The other twosamples each contained a peptoid polymer at 9 mM. One of the peptoidpolymer samples contained the peptoid polymer called “Compound 1,” whilethe other sample contained the peptoid polymer called “Compound 10.” Thesequences of the peptoid polymers are as follows:

Compound 1: Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb; Compound 10:Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp.

The chemical structures for these compounds are provided in Table 2.

After sample preparation, all samples were slow cooled and incubated at−20° C. on a Peltier cooled plate. After one hour, samples were removedand immediately photographed using a digital camera attached to a 180×Stereo Zoom microscope (FIG. 2A). The water and EG samples showedsignificant ice crystal formation, although the EG sample showed lessice formation than the water-only sample. In contrast, neither of thesamples containing the peptoid polymer compounds exhibited significantice crystal formation. Normalized data is presented in FIG. 2B. Of note,the EG sample, containing a CPA concentration that was about 18 timeshigher than the peptoid sample concentrations, still exhibitedsignificant ice formation whereas the peptoid samples did not.

Crystallographic x-Ray Diffraction Assays

In order to increase the throughput of library analysis, acrystallographic x-ray diffraction (XRD) technique was used to evaluateice crystal formation. For these experiments, the compounds named“Compound 2,” “Compound 8,” “Compound 10,” “Compound 11,” “Compound 12,”“Compound 13,” and “Compound 58” were tested. Compounds 2, 8, 10, 11,12, and 13 are peptoid polymers, the structures of which are provided inTable 2. Compound 58 is a peptoid-peptide hybrid, the structure of whichis provided in Table 11. Compound 58 is similar to Compound 12, exceptthat an arginine amino acid has been appended to the N-terminal end.

For these experiments, EG concentrations between 15% and 30% (v/v) wereused. Typically, EG, DMSO, and other cryoprotectants are used during XRDsample analysis at concentrations of 35-40% (v/v) to vitrify solutionsand avoid diffraction interference from ice crystals. Concentrations of1 and 5 mg/mL of the peptoid and peptoid-peptide compounds were used.FIGS. 3A, 3B, and 3C illustrate exemplary XRD data under conditions ofcomplete vitrification, partial vitrification with the presence of cubicice, and freezing (cubic ice crystals), respectively. XRD data forCompounds 8, 10, 11, 12, 13, and 58 is provided in FIGS. 4A-4G and FIGS.5A-5G. FIG. 3D provides ice rings scores for a variety of EGconcentrations and two concentrations of Compounds 2, 8, and 12.

Several mixtures of the testing solution sample sets showed a stronganti-icing effect. FIG. 3D shows the experimental results of somepeptoid polymer solutions compared to EG. “IceRing1” and “IceRing2”refer to ice formation scores, which range between 0 (no ice formation)and 15 (large ice formation). Compounds 2, 12, and 8 and otherssignificantly reduced necessary EG concentrations while preventing iceformation.

The sample containing Compound 12 at a concentration of 5 mg/mL (0.5%(w/v)) and EG at a concentration of 17.5% (v/v) in water was ice-freeafter flash freezing. This particular mixture was found to completelyeliminate all ice formation over multiple trials of flash freezing in astream of liquid nitrogen vapor (FIG. 3A), and vastly outperformed astandard solution of 30% EG (FIG. 3B). In the figures, black spots andrings represent ice crystals. In comparison to EG at the same molarconcentration, this anti-icing effect is 500 times stronger and, withoutbeing bound by any particular theory, suggests a non-colligativemechanism for anti-icing, which is the mechanism used by naturalantifreeze proteins.

Larger Volume Assays

In order to test the usefulness of compositions of the present inventionat larger scales, experiments were performed using solution volumes thatare similar to volumes used for standard egg and stem cell preservation.For these experiments, two samples, one containing 22.5% EG and bufferonly, and another containing 22.5% EG and 5 mg/ml (0.5% w/v) of Compound12 and buffer, were flash frozen in liquid nitrogen. As shown in FIG.6A, the Compound 12 solution showed complete vitrification with no iceformation immediately after removal from liquid nitrogen, while thecontrol solution had clearly been frozen, yielding a mass of white icecrystals. The rewarming of the solutions in a 37° C. water bath led toan unexpected and beneficial result. The Compound 12 solution bypasseddevitrification in less than 2 seconds upon rewarming (FIG. 6B, right),whereas chunks of ice were seen floating in the control sample (FIG. 6B,left) after 20 seconds. Condensation was seen on each of the tubesbecause the tubes were actually still much below room temperature. Thisresult shows that Compound 12 acts as an active de-icer during thawing.

Furthermore, after leaving the 100 μL samples in a −20° C. freezerovernight, the Compound 12 solution was found to be unfrozen (FIG. 6C,right). This result shows that compositions of the present inventionprovide the ability to preserve samples at below 0° C. temperatures forlong periods of time without any ice formation. Furthermore, theseexperiments show that ice-free conditions can be reached withhypothermic cryopreservation, or by the supercooling method, at −20° C.as well as near vitrification to −80° C. by incorporating compounds ofthe present invention to significantly reduce the critical concentrationof penetrating CPAs and mitigate cryopreservation toxicity.

As shown here, a formulation of Compound 12 was found to prevent iceformation during vitrification in sub-milliliter volumes. In fact, thesolutions were able to remain completely unfrozen at −20° C. and werealso able to vitrify when flash frozen at −196° C. Currently, standardhuman egg cell preservation techniques for in vitro fertilization arelimited to solution volumes of less than 5 uL (often 0.5 to 2.5 μL)while using 50% or greater cryoprotectant concentrations. Thus, Compound12 was able to prevent ice formation in a practical volume, withexceedingly less cryoprotectant, which makes it useful, for example, forpreserving human oocytes for in vitro fertilization.

Example 2. Cytotoxicity and Cryopreservation Screening

This example shows that compositions described herein have little to nocell toxicity and can achieve superior cryopreservation when compared toexisting compounds, while reducing the necessary amount of CPAs and thusreducing CPA-associated toxicity.

Cytotoxicity Assays

In order to demonstrate the safety of cryoprotectant compositions of thepresent invention, a high-throughput cell-based cytotoxicity assay wasdeveloped utilizing the HEK 293 cell line, which is a sturdy and robuststem cell line grown from human embryonic kidney cells in tissueculture.

A Tecan Genesis Robotic Workstation was used to prepare solutions in 96-and 384-well plates. Solutions contained culture media, buffers, acryoprotectant composition of the present invention (Compound 12) orDMSO. Solutions were adjusted to the desired pH. Serial dilutions wereperformed to obtain solutions containing various concentrations ofCompound 12 and DMSO. Control experiments were performed using onlyculture media.

For these experiments, cells were seeded at low density (i.e., 10%confluence), exposed to solutions containing Compound 12 or DMSO, andplaced in a 37° C. incubator. The cells were allowed to grow untilcontrol cells that were treated only with empty vehicle approached 70%confluence (typically about 3 to 5 days). Assessment for compoundcytotoxicity was via MTT assay.

As can be seen in FIG. 7 , the toxicity of Compound 12 did notsignificantly deviate from the that of culture media alone when analyzedby MTT assay. On the other hand, DMSO did not allow for warm survivalfor an extended period of time at any concentration above 0.5% (v/v).Notably, Compound 12 did not show toxicity at the concentrations inwhich it can prevent ice formation in a non-biological sample (0.5% w/v)and did not show significant toxicity at concentrations four timesgreater than this concentration, either.

These results show that compositions of the present invention wereeffective at ice-prevention even at concentrations where DMSO toxicitysignificantly reduced cell survival.

Cryopreservation Assays

Initial cryopreservation assays were performed using very simplesolutions, with and without the addition of Compound 12, in order tominimize confounding outside factors. For this first set of experiments,two sample solutions were prepared. The first sample solution containedsimple buffer and ethylene glycol (EG) at a concentration of 22.5%(v/v), and the second sample solution contained simple buffer, EG (22.5%(v/v)), and 5 mg/mL (0.5% (w/v)) of Compound 12.

HEK 293 cells were grown until 70% confluent, then treated with trypsinto remove adhesion proteins and yield free floating cells. Cells werecounted using a hemocytometer and sample cell concentrations wereadjusted to final concentrations of 10,000 cells per microliter. Cellswere then compressed into tight pellets by centrifugation, and eachsample was subsequently mixed with 20 μL of one of the sample solutions.Samples were then flash frozen by immersion in liquid nitrogen, followedby rewarming in a 37° C. water bath. After the freeze-thaw process,cells were suspended in a 400× volume of culture media for recovery. Thepositive control sample was treated with culture media at 37° C. and notsubjected to the freeze-thaw process. The negative control sample wastreated with culture media only during the freeze-thaw process. Afterrecovery, cells were stained with Calcein AM for 30 minutes and cellviability was measured using a fluorescence plate reader.

As shown in FIG. 8 , the addition of Compound 12 greatly improved cellsurvival and demonstrated the ability of this compound to cryopreservecells. It was observed that the sample containing Compound 12 achievedcomplete vitrification without ice formation during the freezingprocess. In addition, the process of devitrification was bypassed muchmore rapidly compared to the sample lacking Compound 12.

A second set of experiments was performed to evaluate thecryopreservation potential of a formulation that contained 5 mg/mL ofCompound 12 plus a mixture of glycols, disaccharides, and a generalbuffer. Post-thaw survival following vitrification in liquid nitrogenwas evaluated as described above. As can be seen in FIG. 9 , theformulation achieved near 100% (i.e., 98%) post-thaw survival of thecells, which was similar to the control group that was not exposed tofreezing treatment. The cell morphologies and florescence signals lookedidentical to the non-frozen controls, which indicated that little damageoccurred to the cells during the experiment.

As part of the second set of experiments, the cryopreservation potentialof the formulation was compared to two known cryopreservation reagents.VS2E is a DMSO-free and serum-free solution containing non-chemicallydefined polymers (see, e.g., Nishigaki et al. Int. J. Dev. Biol.55:3015-311 (2011)), and M22 is an organ vitrification solutionavailable from 21^(st) Century Medicine. FIG. 9 shows that theformulation containing Compound 12 achieved superior cryopreservation,as cell survival was 72% and 51% for VS2E and M22, respectively. Itshould be noted that for the M22 sample, background fluorescence mayhave skewed this result, as a count of live cells in the image suggestedthat far fewer than 51% of the cells had survived.

The compositions of the present invention were highly effective atpreventing ice formation in solutions containing significantly reducedethylene glycol. In particular, low concentrations of the compositions(e.g., 0.5% (w/v)) were sufficient to block ice growth duringvitrification and to keep solutions in a liquid, ice-free state on the20 uL scale, which is a scale that is useful for the preservation ofvarious types of cells.

In summary, these results show that compositions of the presentinvention can achieve superior cryopreservation and reduce the necessaryamount of CPAs, thus reducing cell toxicity that is associated withCPAs. The superior properties of the compositions of the presentinvention are especially useful for the treatment of particularlysensitive cell lines and/or when cells need to be cultured for longertime periods.

Example 3. Supercooling of Cells for Extended Time Periods

This example shows that successful cell preservation using thesupercooling formulas and methods described herein is feasible forextended time periods.

Transporting preserved cells and tissue from lab to patient at highsub-zero temperature could result in: 1) increased cell survival andprolonged storage period (beyond 24 hrs) compared to 4° C. storage; (2)reduced transport cost compared to cryopreservation by avoiding the useof LN2; and (3) avoidance of ice damage that occurs during water phasetransitions in deep cooling storage protocols (e.g., −80° C. and −196°C.). Traditional penetrating CPAs are not able to reach coolertemperatures beyond −6° C. during supercooled storage due to adversetoxicity. Storage at lower temperatures can provide longer metabolicsuspension for longer cell storage periods and can also achieve the bestpractical use with integration in cold chain infrastructure for −20° C.cooling/shipping.

Supercooling Preservation Protocol

We compared various formulas using HEK293 and K562 cells. Briefly, 100μL of cells (0.5×10⁶ cells/mL) were suspended in test cryoprotectant (orin media for the non-frozen control) in replicates. Test tubes wereplaced on a shelf in the −20° C. freezer and removed at designated timepoints. Samples were warmed in a 37° C. water bath and 10 μL of each wasaliquoted into recovery wells containing 500 uL DMEM+10% FBS and placedin an incubator (37° C., 5% CO₂) for 16 hours to allow cells undergoingapoptosis to pass. Cells were either stained with alamarBlue® or datawas acquired by flow cytometry to improve comparison to non-frozen cellsat the same growth stage as those that were subjected to cooling andwarming protocols. Cell counts were normalized to non-frozen arms.

Samples were either placed directly in the −20° C. freezer in cryovialsor more precisely controlled by placing into a Mr. Frosty, which is anisopropanol containing device used to control the rate of cooling acryovial to ˜1° C./min (when placed in a −80° C. freezer). Theappearance of each tube at −20° C. was examined at each of the testintervals after cooling to −20° C. and noted as either liquid,semi-solid, or crystalline, which was designed to inform correlationbetween ice formation and cell survival.

Results

Cell survival was demonstrated with formulas for 1- and 3-day storage at−20° C. Increased cell survival was observed for formulas that exhibitedcomparatively less or no crystal formation. However, a bettercorrelation for increased cell survival was noticed for formulascontaining active peptoid. The formulas also provided consistent cellsurvival rates after day 1 and day 3, whereas DMSO and media show adrastic difference between time points.

The formulas were further tuned with cross experiments of various basebuffers, small molecule CPA concentrations, and select cellularprotection agents. Protocol improvements examined cells preservation at(−20° C.) after 3, 24, 48, 72, and 120 hours along with many otherformulation comparisons. We also expanded our cell models to includeK562 cells as a representative of a non-adherent blood cell and Jurkatcells.

FIGS. 10A and 10B show that the methods of the present invention enabledsupercooling of cells at −20° C. Formulas containing peptoid polymerssuch as Compound 12 had the unique ability to preserve cells at −20° C.with significant viability after 72 hrs under supercooled conditions. Incontrast, HTK buffer at its proposed shipping/working temperature (4°C.), an FDA approved organ preservation solution designed to ship humanorgans for transplant, only maintained cell survival less than 24 hrs at4° C.

Example 4. Supercooling of Primary Genitourinary Cells and Tissues forTransplant

This example shows the successful preservation of primary genitourinary(GU) cells using the supercooling formulas and methods described herein.

Examples of human GU cell types include BJ (human fibroblast foreskin),PC-3 (human prostate adenocarcinoma), and SK-OV-3 (human ovaryadenocarcinoma). Examples of primary GU cell types include primarycorpus cavernosum (CC) cells such as smooth muscle and endothelialcells. In penile tissue, CC endothelial and smooth muscle cells providethe basic form and function. Cell preservation experiments can beperformed using GU explants.

Endothelial Cells

Briefly, corpus cavernosum (CC) tissue was harvested, processed,digested and filtered to yield single endothelial cells which were thenresuspended in cell medium followed by cultivation for 5 days. See,Chung et al., Korean Journal of Urology, 53(8):556-563 (2012). Confluentadherent endothelial cells were further processed, resuspended in RPMImedium and filtered through a 70 μm nylon mesh. The cells were thenincubated with biotinylated anti-rat CD146 antibody at 20° C. for 30min. After washing, cells were resuspended in buffer and were incubatedwith streptavidin coated magnetic beads. Magnetically labelled CD146cells were isolated using a magnetic column. See, Weber et al., PediatrRes, 70(3):236-41 (2011). The eluate was resuspended in EC medium andcultured.

Smooth Muscle Cells

Cavernosal tissue was washed, processed and placed in a minimal volumeof supplemented DMEM at 37° C. in a humidified atmosphere of 95% air and5% CO₂. See, Pilatz et al., European Urology, 47(5):710-719 (2005).After further processing, cells migrated out of the explants (4-10days), the explants were removed, and the cells were allowed to achieveconfluence with D-valine in culture medium to control the outgrowth offibroblast cells without affecting smooth muscle cell morphology. Cellswere examined for alpha smooth muscle actin (α-SMA) expression byimmunostaining and fluorescence microscopy.

Supercooling Preservation Protocol

A small amount of CC endothelial and smooth muscle cells were used atapproximately 70% average confluence. Smaller volumes were necessitatedfor this experiment, so 1 μL of a prepared cell slurry was suspended in19 μL (250,000 cells/mL) of several formulas in a Biorad 96-well PCRplate. The well plate was placed in a −20° C. freezer equipped with aPeltier plate for temperature stability and cells were sampled atdesignated time points. A 24-well tissue culture plate containing 1 mLof either Medium 199 with 20% FBS (primary cells) or DMEM+10% FBS(HEK293 control) were pre-equilibrated at 37° C. with 5% CO₂ for 1 hour.The cryopreserved cell solutions (20 μL) were pipetted into 24-welltissue culture plate and allowed to recover overnight followed bysurvival assay with calcein AM 24 hours post-warm. HEK293 cells servedas an external cell counting standard and quantitated in high fidelity(R²>98).

Results

Formulas mixed with primary cells stored at −20° C. appeared unfrozen ateach time point after supercooling to −20° C. FIG. 11 shows that HTKsolution failed to protect CC endothelial cells at −20° C., resulting inzero cell survival after 48 hrs, probably due to solid ice formationduring the preservation. Similar results were obtained for CC smoothmuscle cells. Importantly, the use of supercooling formula XT-SC5resulted in higher survival of primary CC endothelial cells and smoothmuscle cells after 48 hrs compared to formulas XT-SC6, XT-SC7, andXT-SC8. XT-SC5 shares the same basic buffer as XT-SC6, XT-SC7, andXT-SC8, but with the addition of 0.5% peptoid Compound 12. All fourformulas contain different concentrations of small molecule CPAs(XT-SC5=XT-SC6, XT-SC7=1.25X, and XT-SC8=1.5X).

The preservation and time course survival study of endothelial cellsreveals that XT-SC5 with peptoid polymer provides exceptional cellsurvival (94%) over a time course of 48 hrs, while formulas withoutpeptoid polymer and with increased CPA concentration exhibit reducedsurvival of endothelial cells over 48 hrs (approximately 20%). Theoverall trend of smooth muscle cell survival is consistent with theresult obtained from the endothelial cell preservation.

Example 5. Supercooling of Genitourinary Cells from Model Cell Line

This example shows the successful preservation of genitourinary (GU)cells from a model human GU cell line using the supercooling formulasand methods described herein.

Cryopreservation experiments were performed at −20° C. Survival,viability, and proliferation were examined. Cells were analyzed byfluorescence plate reading assays.

The cell line SK-OV-3 (human ovary adenocarcinoma) was preserved as amodel human genitourinary cell type at −20° C. in variouscryopreservation solutions containing a peptoid polymer (e.g., Compounds1-8). Each solution had 4 identical replicas at 5 time points. At eachtime point, the cell numbers were quantitated with an external standard.

Briefly, 100 μL of cells (0.5×10⁶ cells/mL) were suspended in testcryoprotectant (or in media for the non-frozen control) in replicates.Test tubes were placed on a shelf in the −20° C. freezer and removed atdesignated time points. Samples were warmed in a 37° C. water bath and10 IA of each was aliquoted into recovery wells containing 500 μLDMEM+10% FBS and placed in an incubator (37° C., 5% CO₂) for 16 hours toallow cells undergoing apoptosis to pass. Cells were stained and cellcounts were normalized.

We compared the survival of cells in formulas that were forced to freezeversus unfrozen formulas. As set forth in Table 12, most of the formulasshowed very high survival over 6 days when they remained unfrozen aftersupercooling to −20° C. (“Liquid”) compared to formulas that were forcedfrozen by crystal seeding with ice crystals (“Frozen”). These resultsdemonstrate that cells which remain in liquid at −20° C. for 6 days havea much higher survival rate and establish that supercooled, non-frozensolutions enhance cell survival under conditions of reduced temperatureand metabolism.

TABLE 12 Day 6 Survival Compound Frozen Liquid 1 18% 83% 2 15% 82% 3  7%20% 4  3% 83% 5 20% 86% 6 16% 91% 7 18% 50% 8 18% 73%

Example 6. Survival and Proliferation Studies on Supercooled Cells

This example shows that cells preserved in accordance with thesupercooling formulas and methods described herein are viable anddemonstrate enhanced survival and proliferation compared to controlsamples without peptoid polymers.

SK-OV-3 Cells

We evaluated cell survival and proliferation following supercooledpreservation of SK-OV-3 cells at −20° C. in several formulas for 3 daysin a solution containing significantly reduced small moleculecryoprotectant (about 5× lower concentration). A comparison was madebetween a cryopreservation formula with 1% Compound 12 (“XT Formula+1%Compound 12”) and XT Formula only, a 10% DMSO formula (standard forcryopreservation), and/or DMEM (base media, negative control). “XTFormula” contains HTK buffer and other components.

FIG. 12 shows that the formula containing a peptoid polymer waseffective at enhancing SK-OV-3 cell survival following the supercoolingpreservation methods described herein. In particular, at least 50% ofthe supercooled cells in the peptoid formula survived at 1 daypost-warming compared to the starting cell number and the number ofcells in the peptoid formula at 1 day post-warming was at least about 2to 4-fold greater compared to the formula without the peptoid polymer orthe DMSO formula.

FIG. 13 shows that the formula containing a peptoid polymer waseffective at enhancing SK-OV-3 cell viability following the supercoolingpreservation methods described herein. In particular, the number ofcells in the peptoid formula at 3 days post-warming was at least about 5to 6-fold greater compared to the formula containing DMSO.

K562 Cells

We evaluated cell proliferation following supercooled preservation ofK562 cells at −20° C. in several formulas after 3, 6, or 7 days ofincubation post-warming. K562 cells were preserved by supercooling for 3days at −20° C. comparing XT Formula with a peptoid polymer (e.g.,Compound 12) to XT Formula only (i.e., no peptoid), 10% DMSO formula(standard for cryopreservation), and DMEM (base media, negativecontrol). Briefly, 200 μL of cells (0.5×10⁶ cells/mL) were suspended intest cryoprotectant (or in media for the non-frozen control) at 4° C.for 10 minutes in cryovials by using aluminum block in replicates. Testtubes were placed on a shelf in the −20° C. freezer and removed atdesignated time points. Samples were warmed in a 37° C. water bath withgentle swirling and 2 μL of each (10,000 cells) was aliquoted intorecovery wells containing 200 μL DMEM+10% FBS and placed in an incubator(37° C., 5% CO₂). Cells were incubated for 3, 6, or 7 days, stained atvarious intervals, and analyzed by plate reader.

FIGS. 14 and 15 show that the formula containing a peptoid polymer waseffective at enhancing K562 cell viability following the supercoolingpreservation methods described herein. In particular, FIG. 14 shows thatthe number of cells in the peptoid formula with Compound 12 at 3 dayspost-warming was at least about 1 to 2-fold greater compared to theformula without the peptoid polymer or the DMSO formula. FIG. 15 showsthat the number of cells in the peptoid formula with Compound 12 at 6days post-warming was at least about 2 to 3-fold greater compared to theformula without the peptoid polymer or the DMSO formula.

FIG. 16 shows that numerous formulas containing various peptoid polymerswere effective at enhancing K562 cell viability following thesupercooling preservation methods described herein. In particular, thenumber of cells in the peptoid formula with Compound 12, 27, 62, 74, or76 at 7 days post-warming was at least about 3 to 4-fold greatercompared to the formula containing DMSO.

V. Exemplary Embodiments

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

1. A method for cryopreserving a population of cells with improved cellviability, the method comprising:

-   -   (a) contacting a population of cells with a peptoid polymer or a        salt thereof comprising one or more polar peptoid monomers; and    -   (b) cooling the population of cells to a temperature of from        0° C. to about −20° C. for a time period of at least about 3        hours to produce a population of supercooled cells,    -   wherein at least about 50% of the population of supercooled        cells survive after warming to above 0° C.

2. The method of embodiment 1, wherein the temperature is from about−10° C. to about −20° C.

3. The method of embodiment 1 or 2, wherein the temperature is about−20° C.

4. The method of any one of embodiments 1 to 3, wherein the time periodis at least about 8 or 16 hours.

5. The method of any one of embodiments 1 to 3, wherein the time periodis from about 2 to about 5 days.

6. The method of embodiment 5, wherein the time period is about 2, 3, 4,or 5 days.

7. The method of any one of embodiments 1 to 3, wherein the time periodis at least about 5 days.

8. The method of any one of embodiments 1 to 7, wherein at least about50% of the population of supercooled cells survive after warming to 37°C.

9. The method of any one of embodiments 1 to 8, wherein at least about60% of the population of supercooled cells survive after warming.

10. The method of any one of embodiments 1 to 8, wherein at least about70% of the population of supercooled cells survive after warming.

11. The method of any one of embodiments 1 to 8, wherein at least about80% of the population of supercooled cells survive after warming.

12. The method of any one of embodiments 1 to 8, wherein at least about90% of the population of supercooled cells survive after warming.

13. The method of any one of embodiments 1 to 12, wherein the improvedcell viability comprises enhanced proliferation of the population ofsupercooled cells that survive after warming compared to a controlpopulation of supercooled cells.

14. The method of embodiment 13, wherein the control population ofsupercooled cells has not been contacted with the peptoid polymer.

15. The method of embodiment 13 or 14, wherein the number of cells inthe population of supercooled cells at about 3 days after warming is atleast about 1-fold greater than the number of cells in the controlpopulation of supercooled cells.

16. The method of embodiment 13 or 14, wherein the number of cells inthe population of supercooled cells at about 6 days after warming is atleast about 2-fold greater than the number of cells in the controlpopulation of supercooled cells.

17. The method of any one of embodiments 1 to 16, wherein the peptoidpolymer is present in an amount sufficient to reduce or inhibit icecrystal formation at the temperature.

18. The method of embodiment 17, wherein the peptoid polymer is presentin amount between about 100 nM and about 1000 mM.

19. The method of any one of embodiments 1 to 18, wherein the populationof cells comprises a tissue or an organ.

20. The method of any one of embodiments 1 to 19, wherein the populationof cells is selected from the group consisting of primary cells, heartcells, liver cells, lung cells, kidney cells, pancreatic cells, gastriccells, intestinal cells, muscle cells, skin cells, neural cells, bloodcells, immune cells, fibroblasts, genitourinary cells, bone cells, stemcells, sperm cells, oocytes, embryonic cells, epithelial cells,endothelial cells, and a combination thereof.

21. The method of any one of embodiments 1 to 20, wherein the methodfurther comprises:

-   -   (c) warming the population of supercooled cells to above 0° C.

22. The method of any one of embodiments 1 to 21, wherein the peptoidpolymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, ormore polar peptoid monomers.

23. The method of any one of embodiments 1 to 22, wherein the polarpeptoid monomers have independently selected side chains comprising ahydroxyl group.

24. The method of embodiment 23, wherein the independently selected sidechains are optionally substituted C₁₋₁₈ hydroxyalkyl groups.

25. The method of embodiment 24, wherein the C₁₋₁₈ hydroxyalkyl groupsare independently selected optionally substituted C₁₋₆ hydroxyalkylgroups.

26. The method of any one of embodiments 1 to 25, wherein the peptoidpolymer is a peptoid-peptide hybrid or a salt thereof comprising thepeptoid polymer and one or more amino acids, wherein the one or moreamino acids are located at one or both ends of the peptoid polymerand/or between one or more peptoid monomers.

27. The method of any one of embodiments 1 to 26, wherein the peptoidpolymer has a structure according to formula (I):

-   -   a tautomer thereof or stereoisomer thereof,    -   wherein:    -   each R¹ is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted alkoxy,        optionally substituted C₁₋₁₈ alkylamino, optionally substituted        C₁₋₁₈ alkylthio, optionally substituted carboxyalkyl, C₃₋₁₀        cycloalkyl, heterocycloalkyl, aryl, heteroaryl, (C₃₋₁₀        cycloalkyl)alkyl, (heterocycloalkyl)alkyl, arylalkyl, and        heteroarylalkyl,    -   wherein at least one instance of R¹ is an optionally substituted        C₁₋₁₈ hydroxyalkyl group, and    -   wherein any of the cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl groups is optionally and independently substituted        with one or more R³ groups;    -   each R² is independently selected from the group consisting of        H, optionally substituted C₁₋₁₈ alkyl, optionally substituted        C₂₋₁₈ alkenyl, optionally substituted C₂₋₁₈ alkynyl, optionally        substituted C₁₋₁₈ hydroxyalkyl, optionally substituted C₁₋₁₈        alkylamino, optionally substituted C₁₋₁₈ alkylthio, and        optionally substituted carboxyalkyl;    -   each R³ is independently selected from the group consisting of        halogen, oxo, thioxo, —OH, —SH, amino, C₁₋₈ alkyl, C₁₋₈        hydroxyalkyl, C₁₋₈ alkylamino, and C₁₋₈ alkylthio;    -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, acetyl, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond; and    -   the subscript n, representing the number of monomers in the        polymer, is between 2 and 50.

28. The method of embodiment 27, wherein at least 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, or more instances of R¹ are independently selectedoptionally substituted C₁₋₁₈ hydroxyalkyl groups.

29. The method of embodiment 28, wherein the C₁₋₁₈ hydroxyalkyl groupsare independently selected optionally substituted C₁₋₆ hydroxyalkylgroups.

30. The method of embodiment 27, wherein each R¹ is independentlyselected from the group consisting of

-   -   wherein:    -   m is between 1 and 8; and    -   R³ is selected from the group consisting of H, C₁₋₈ alkyl,        halogen, hydroxyl, thiol, nitro, amine, oxo, and thioxo.

31. The method of embodiment 30, wherein one or more R¹ has a structureaccording to Rib:

32. The method of embodiment 27, wherein each R¹ is independentlyselected from the group consisting of

33. The method of any one of embodiments 27 to 32, wherein each instanceof R² is H.

34. The method of any one of embodiments 27 to 33, wherein n is between6 and 25.

35. The method of any one of embodiments 27 to 33, wherein n is between6 and 20.

36. The method of any one of embodiments 27 to 33, wherein n is between10 and 25.

37. The method of any one of embodiments 27 to 36, wherein X is selectedfrom the group consisting of H, C₁₋₈ alkyl, and C₁₋₈ acyl; and Y isselected from the group consisting of —OH and amino.

38. The method of any one of embodiments 1 to 26, wherein the peptoidpolymer comprises subunits comprising one or more first hydrophobicpeptoid monomers H and one or more first polar peptoid monomers Parranged such that the peptoid polymer has the sequence [H_(a)P_(b)]_(n)or [P_(b)H_(a)]_(n), wherein:

-   -   the subscript a, representing the number of consecutive first        hydrophobic peptoid monomers within a subunit, is between 1 and        10;    -   the subscript b, representing the number of consecutive first        polar peptoid monomers within a subunit, is between 1 and 10;        and    -   the subscript n, representing the number of subunits within the        peptoid polymer, is between 2 and 50.

39. The method of embodiment 38, further comprising substituents X and Ysuch that the peptoid polymer has the sequence X—[H_(a)P_(b)]_(n)—Y orX—[P_(b)H_(a)]_(n)—Y, wherein:

-   -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond.

40. The method of embodiment 38 or 39, wherein the subunits furthercomprise a second hydrophobic peptoid monomer and/or a second polarpeptoid monomer such that the peptoid polymer has the sequence[H_(a)P_(b)H_(c)Pci]_(n) or [P_(b)H_(a)P_(d)H_(c)]_(n), wherein:

-   -   the subscript c, representing the number of consecutive second        hydrophobic peptoid monomers within a subunit, is between 0 and        10;    -   the subscript d, representing the number of consecutive second        polar peptoid monomers within a subunit, is between 0 and 10;        and    -   both c and d are not 0.

41. The method of embodiment 40, further comprising substituents X and Ysuch that the peptoid polymer has the sequenceX—[H_(a)P_(b)H_(c)P_(d)]_(n)—Y or X—[P_(b)H_(a)P_(d)H_(c)]_(n)—Y,wherein:

-   -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond.

42. The method of any one of embodiments 38 to 41, further comprising asequence Z that comprises one or more hydrophobic peptoid monomersand/or one or more polar peptoid monomers, wherein Z is located beforethe first subunit, after the last subunit, and/or between one or moresubunits.

43. The method of any one of embodiments 1 to 26, wherein the peptoidpolymer comprises:

-   -   (a) subunits comprising two first hydrophobic peptoid monomers H        and two first polar peptoid monomers P, and    -   (b) two second hydrophobic peptoid monomers located at the        C-terminal end of the peptoid polymer,    -   arranged such that the peptoid polymer has the sequence        [H₂P₂]_(n)H₂ or [P₂H₂]_(n)H₂, wherein the subscript n,        representing the number of subunits within the peptoid polymer,        is between 1 and 50.

44. The method of embodiment 43, wherein the peptoid polymer furthercomprises substituents X and Y such that the peptoid polymer has thesequence X—[H₂P₂]_(n)H₂—Y or X—[P₂H₂]_(n)H₂—Y, wherein:

-   -   X and Y are independently selected from the group consisting of        H, optionally substituted C₁₋₈ alkyl, optionally substituted        C₁₋₈ acyl, optionally substituted C₁₋₈ alkylamino, —OH, —SH,        —NH₂, carboxy, optionally substituted C₁₋₈ hydroxyalkyl,        optionally substituted C₁₋₈ alkylamino, optionally substituted        C₂₋₈ alkylthio, optionally substituted C₁₋₈ carboxyalkyl, and        halogen, or    -   alternatively X and Y are taken together to form a covalent        bond.

45. The method of embodiment 43 or 44, wherein n is between 1 and 10.

46. The method of any one of embodiments 38 to 45, wherein the firstand/or second hydrophobic peptoid monomers are independently selectedfrom the group consisting of

-   -   wherein the subscript m is the number of repeat units and is        between 1 and 10.

47. The method of any one of embodiments 38 to 46, wherein the peptoidpolymer comprises a polar peptoid monomer having a side chain thatcomprises a hydroxyl group.

48. The method of any one of embodiments 38 to 47, wherein the firstand/or second polar peptoid monomers are independently selected from thegroup consisting of

wherein the subscript m is the number of repeat units and is between 1and 10.

49. The method of any one of embodiments 38 to 48, wherein each of thefirst and/or second polar peptoid monomers comprise a side chain that isindependently selected from the group consisting of (C₁₋₆ alkoxy)(C₁₋₆alkylene), oligo(ethylene glycol), (4-to 10-memberedheterocycloalkyl)(C₁₋₆ alkylene), and (5- to 10-memberedheteroaryl)(C₁₋₆ alkylene).

50. The method of embodiment 49, wherein (4- to 10-memberedheterocycloalkyl)(C₁₋₆ alkylene) comprises a 4-6 membered heterocyclicring, wherein at least one member is selected from the group consistingof 0 and N.

51. The method of embodiment 49 or 50, wherein (4- to 10-memberedheterocycloalkyl)(C₁₋₆ alkylene) comprises a tetrahydrofuranyl oroxopyrrolidinyl moiety.

52. The method of embodiment 51, wherein the peptoid polymer comprises

53. The method of embodiment 52, wherein all of the polar peptoidmonomers are

54. The method of embodiment 49, wherein (5- to 10-memberedheteroaryl)(C₁₋₆ alkylene) comprises a 5-6 membered aromatic ring,wherein at least one ring member is selected from the group consistingof 0 and N.

55. The method of embodiment 49 or 54, wherein (5- to 10-memberedheteroaryl)(C₁₋₆ alkylene) comprises a furanyl moiety.

56. The method of embodiment 55, wherein the peptoid polymer comprises

57. The method of embodiment 49, wherein the side chain comprises amethoxyethyl group.

58. The method of embodiment 57, wherein the peptoid polymer comprises

59. The method of embodiment 49, wherein the side chain comprises anoligo(ethylene glycol) moiety.

60. The method of embodiment 59, wherein the oligo(ethylene glycol)moiety is a 2-(2-(2-methoxyethoxy)ethoxy)ethyl moiety.

61. The method of embodiment 60, wherein the peptoid polymer comprises

62. The method of any one of embodiments 38 to 42 or 46 to 61, wherein nis between 2 and 10.

63. The method of any one of embodiments 38 to 42 or 46 to 62, where ais between 1 and 5.

64. The method of any one of embodiments 38 to 42 or 46 to 63, wherein bis between 1 and 5.

65. The method of embodiment 63 or 64, wherein a is between 1 and 3 andb is between 1 and 3.

66. The method of any one of embodiments 40 to 42 or 46 to 65, wherein cis between 0 and 5.

67. The method of any one of embodiments 40 to 42 or 46 to 66, wherein dis between 0 and 5.

68. The method of any one of embodiments 38 to 67, wherein about 10, 20,30, 40, 50, 60, 70, 80, or 90 percent of the peptoid monomers arehydrophobic.

69. The method of any one of embodiments 1 to 68, wherein the peptoidpolymer salt is selected from the group consisting of a hydrochloridesalt, acetate salt, sulfate salt, phosphate salt, maleate salt, citratesalt, mesylate salt, nitrate salt, tartrate salt, gluconate salt, and acombination thereof.

70. The method of any one of embodiments 1 to 69, wherein the peptoidpolymer is formulated in a cryoprotectant solution.

71. The method of embodiment 70, wherein the cryoprotectant solutionfurther comprises a compound selected from the group consisting of anionic species, a penetrating cryoprotectant, a non-penetratingcryoprotectant, an antioxidant, a cell membrane stabilizing compound, anaquaporin or other channel forming compound, an alcohol, a sugar, asugar derivative, a nonionic surfactant, a protein, dimethyl sulfoxide(DMSO), polyethylene glycol (PEG), polypropylene glycol (PPG), Ficoll®,polyvinylpyrrolidone, polyvinyl alcohol, hyaluronan, formamide, anatural or synthetic hydrogel, and a combination thereof.

72. A population of supercooled cells with improved cell viabilityproduced by the method of any one of embodiments 1 to 71.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described. All publications, patents, and patentapplications cited in this specification are herein incorporated byreference as if each individual publication, patent, or patentapplication were specifically and individually indicated to beincorporated by reference.

VI. Informal Sequence Listing Sequence NotesNsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nsb Compound 1Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp-Nhp Compound 10Nep-Nep-Xaa-Xaa-Xaa-Xaa-Nep-Nep-Nep-Nep-Nme-Nme Peptoid-Peptide HybridNme-Nme-Xaa-Nme-Nme-Nme-Nme-Nhp-Nhp-Nsb-Xaa- Peptoid-Peptide HybridNme-Nme-Xaa-Nme-Nme-Nme Nme-Nme-Xaa-Nme-Nme-Nme-Nme-Nme-Nme-Nme-Peptoid-Peptide Hybrid Xaa-XaaArg-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Peptoid-Peptide Hybrid(Compound 58) Nsb-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp-Nsb-Nhp Compound 6Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 12Nsb-Nhp-Nhp-Nhp-Nhp-Nhp-Nsb-Nhp-Nhp-Nhp Compound 8Nsb-Nsb-Nsb-Nhp-Nhp-Nhp-Nsb-Nsb-Nsb-Nhp Compound 2Nib-Nib-Nhp-Nhp-Nib-Nib-Nhp-Nhp-Nib-Nib Compound 25Nbu-Nbu-Nhp-Nhp-Nbu-Nbu-Nhp-Nhp-Nbu-Nbu Compound 26Npr-Npr-Nhp-Nhp-Npr-Npr-Nhp-Nhp-Npr-Npr Compound 27Nip-Nip-Nhp-Nhp-Nip-Nip-Nhp-Nhp-Nip-Nip Compound 28Nmb-Nmb-Nhp-Nhp-Nmb-Nmb-Nhp-Nhp-Nmb-Nmb Compound 59Ac-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 60 (Compound 12 withacetylated N-terminus) Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-COOHCompound 61 (Compound 12 with carboxy C-terminus)Nsb-Nsb-Nmo-Nmo-Nsb-Nsb-Nmo-Nmo-Nsb-Nsb Compound 62Nsb-Nsb-Ntf-Ntf-Nsb-Nsb-Ntf-Ntf-Nsb-Nsb Compound 63Nme-Nme-Nhp-Nhp-Nme-Nme-Nhp-Nhp-Nme-Nme Compound 64Nbr-Nbr-Nhe-Nhe-Nbr-Nbr-Nhe-Nhe-Nbr-Nbr Compound 65Npr-Npr-Nrh-Nrh-Npr-Npr-Nrh-Nrh-Npr-Npr Compound 66Nsb-Nsb-N3p-N3p-Nsb-Nsb-N3p-N3p-Nsb-Nsb Compound 67Nsb-Nsb-Ndh-Ndh-Nsb-Nsb-Ndh-Ndh-Nsb-Nsb Compound 68Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nsb-Nsb-Nsb Compound 69Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nsb-Nhp-Nsb-Nsb Compound 70Nsb-Nsb-Nhp-Nsb-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 71Nsb-Nsb-Nsb-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 72Nsb-Nsb-Nff-Nff-Nsb-Nsb-Nff-Nff-Nsb-Nsb Compound 73Nsb-Nsb-Nhe-Nhe-Nsb-Nsb-Nhe-Nhe-Nsb-Nsb Compound 74Nsb-Nsb-Nyp-Nyp-Nsb-Nsb-Nyp-Nyp-Nsb-Nsb Compound 75Nsb-Nsb-Nop-Nop-Nsb-Nsb-Nop-Nop-Nsb-Nsb Compound 76Nbr-Nbr-Nrh-Nrh-Nbr-Nbr-Nrh-Nrh-Nbr-Nbr Compound 77Nbr-Nbr-Nsh-Nsh-Nbr-Nbr-Nsh-Nsh-Nbr-Nbr Compound 78Nsb-Nsb-Ndp-Ndp-Nsb-Nsb-Ndp-Ndp-Nsb-Nsb Compound 79Nrh-Nrh-Nrh-Nrh-Nrh-Nrh-Nrh-Nrh-Nrh-Nrh Compound 80Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Compound 81 Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-NsbCompound 82 Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp- Compound 83Nsb-Nsb Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp- Compound 84Nsb-Nsb-Nhp-Nhp-Nsb-Nsb Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Compound 85 Nsb-Nsb-Nhp-Nhp-Nsb-Nsb-Nhp-Nhp-Nsb-NsbNbr-Nbr-Nop-Nop-Nbr-Nbr-Nop-Nop-Nbr-Nbr Compound 86Nbs-Nbs-Nop-Nop-Nbs-Nbs-Nop-Nop-Nbs-Nbs Compound 87Nbs-Nbs-Nrh-Nrh-Nbs-Nbs-Nrh-Nrh-Nbs-Nbs Compound 88Nbs-Nbs-Nsh-Nsh-Nbs-Nbs-Nsh-Nsh-Nbs-Nbs Compound 89Nbr-Nbs-Nrh-Nrh-Nbr-Nbs-Nrh-Nrh-Nbr-Nbs Compound 90Nbs-Nbr-Nrh-Nrh-Nbs-Nbr-Nrh-Nrh-Nbs-Nbr Compound 91Nbr-Nbs-Nsh-Nsh-Nbr-Nbs-Nsh-Nsh-Nbr-Nbs Compound 92Nbs-Nbr-Nsh-Nsh-Nbs-Nbr-Nsh-Nsh-Nbs-Nbr Compound 93Nbr-Nbr-Nrh-Nsh-Nbr-Nbr-Nrh-Nsh-Nbr-Nbr Compound 94Nbr-Nbr-Nsh-Nrh-Nbr-Nbr-Nsh-Nrh-Nbr-Nbr Compound 95Nbs-Nbs-Nrh-Nsh-Nbs-Nbs-Nrh-Nsh-Nbs-Nbs Compound 96Nbs-Nbs-Nsh-Nrh-Nbs-Nbs-Nsh-Nrh-Nbs-Nbs Compound 97Nbr-Nbs-Nrh-Nsh-Nbr-Nbs-Nrh-Nsh-Nbr-Nbs Compound 98Nbr-Nbs-Nsh-Nrh-Nbr-Nbs-Nsh-Nrh-Nbr-Nbs Compound 99Nbs-Nbr-Nrh-Nsh-Nbs-Nbr-Nrh-Nsh-Nbs-Nbr Compound 100Nbs-Nbr-Nsh-Nrh-Nbs-Nbr-Nsh-Nrh-Nbs-Nbr Compound 101

1-72. (canceled)
 73. A method for preserving a biological sample or biological macromolecule, the method comprising: (a) contacting a biological sample or biological macromolecule with a solution comprising a peptoid polymer or a salt thereof comprising one or more polar peptoid monomers; and (b) cooling the solution to a temperature of from 0° C. to about −20° C. for a time period of at least about 3 hours to produce a preserved biological sample or preserved biological macromolecule, wherein the peptoid polymer is present in an amount sufficient to reduce or inhibit ice crystal formation at the temperature, and wherein the solution comprising the preserved biological sample or preserved biological macromolecule is unfrozen at the temperature.
 74. The method of claim 73, wherein the biological sample comprises a tissue, organ, or cells.
 75. The method of claim 74, wherein the tissue is a bioengineered tissue.
 76. The method of claim 74, wherein the tissue or organ is selected from the group consisting of heart, liver, lung, kidney, pancreas, intestine, thymus, cornea, bone marrow, organoids, and a combination thereof.
 77. The method of claim 74, wherein the cells are selected from the group consisting of primary cells, heart cells, liver cells, lung cells, kidney cells, pancreatic cells, gastric cells, intestinal cells, muscle cells, skin cells, neural cells, blood cells, immune cells, fibroblasts, genitourinary cells, bone cells, stem cells, sperm cells, oocytes, embryonic cells, epithelial cells, endothelial cells, and a combination thereof.
 78. The method of claim 73, wherein the biological macromolecule is selected from the group consisting of a nucleic acid, an amino acid, a protein, an isolated protein, a peptide, a lipid, a composite structure, and a combination thereof.
 79. The method of claim 78, wherein the biological macromolecule is a nucleic acid.
 80. The method of claim 79, wherein the nucleic acid is DNA or RNA.
 81. The method of claim 73, wherein the preserved biological sample or preserved biological macromolecule is in a liquid, ice-free suspension at the temperature.
 82. The method of claim 73, wherein the temperature is from about −5° C. to about −20° C.
 83. The method of claim 73, wherein the time period is at least about 8 or 16 hours.
 84. The method of claim 73, wherein the time period is from about 2 to about 5 days.
 85. The method of claim 73, wherein the time period is at least about 72 hours.
 86. The method of claim 73, wherein the peptoid polymer comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more polar peptoid monomers.
 87. The method of claim 73, wherein the one or more polar peptoid monomers have independently selected side chains comprising a hydroxyl group.
 88. The method of claim 87, wherein the independently selected side chains are optionally substituted C₁₋₁₈ hydroxyalkyl groups.
 89. The method of claim 88, wherein the C₁₋₁₈ hydroxyalkyl groups are independently selected optionally substituted C₁₋₆ hydroxyalkyl groups.
 90. The method of claim 73, wherein at least 2 of the polar peptoid monomers have a side chain that comprises an independently selected optionally substituted C₁₋₁₈ hydroxyalkyl group.
 91. The method of claim 90, wherein the C₁₋₁₈ hydroxyalkyl group is an independently selected optionally substituted C₁₋₆ hydroxyalkyl group.
 92. The method of claim 73, wherein the peptoid polymer is a peptoid-peptide hybrid or a salt thereof comprising the peptoid polymer and one or more amino acids, wherein the one or more amino acids are located at one or both ends of the peptoid polymer and/or between one or more peptoid monomers.
 93. The method of claim 73, wherein the peptoid polymer salt is selected from the group consisting of a hydrochloride salt, acetate salt, sulfate salt, phosphate salt, maleate salt, citrate salt, mesylate salt, nitrate salt, tartrate salt, gluconate salt, and a combination thereof. 